Christian Wilms

Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

Spino‐dendritic cross‐talk in rodent Purkinje neurons mediated by endogenous Ca2+‐binding proteins

The range of actions of the second messenger Ca2+ is a key determinant of neuronal excitability and plasticity. For dendritic spines, there is on‐going debate regarding how diffusional efflux of Ca2+ affects spine signalling. However, the consequences of spino‐dendritic coupling for dendritic Ca2+ homeostasis and downstream signalling cascades have not been explored to date. We addressed this question by four‐dimensional computer simulations, which were based on Ca2+‐imaging data from mice that either express or lack distinct endogenous Ca2+‐binding proteins. Our simulations revealed that single active spines do not affect dendritic Ca2+ signalling. Neighbouring, coactive spines, however, induce sizeable increases in dendritic [Ca2+]i when they process slow synaptic Ca2+ signals, such as those implicated in the induction of long‐term plasticity. This spino‐dendritic coupling is mediated by buffered diffusion, specifically by diffusing calbindin‐bound Ca2+. This represents a central mechanism for activating calmodulin in dendritic shafts and therefore a novel form of signal integration in spiny dendrites.

Reading out a spatiotemporal population code by imaging neighbouring parallel fibre axons in vivo

The spatiotemporal pattern of synaptic inputs to the dendritic tree is crucial for synaptic integration and plasticity. However, it is not known if input patterns driven by sensory stimuli are structured or random. Here we investigate the spatial patterning of synaptic inputs by directly monitoring presynaptic activity in the intact mouse brain on the micron scale. Using in vivo calcium imaging of multiple neighbouring cerebellar parallel fibre axons, we find evidence for clustered patterns of axonal activity during sensory processing. The clustered parallel fibre input we observe is ideally suited for driving dendritic spikes, postsynaptic calcium signalling, and synaptic plasticity in downstream Purkinje cells, and is thus likely to be a major feature of cerebellar function during sensory processing.

SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency.

Contemporary research aims to understand biological processes not only by identifying participating proteins, but also by characterizing the dynamics of their interactions. Because Försters Resonance Energy Transfer (FRET) is invaluable for the latter undertaking, its usage is steadily increasing. However, FRET measurements are notoriously error-prone, especially when its inherent efficiency is low, a not uncommon situation. Furthermore, many FRET methods are either difficult to implement, are not appropriate for observation of cellular dynamics, or report instrument-specific indices that hamper communication of results within the scientific community. We present here a novel comprehensive spectral methodology, SpRET, which substantially increases both the reliability and sensitivity of FRET microscopy, even under unfavorable conditions such as weak fluorescence or the presence of noise. While SpRET overcomes common pitfalls such as interchannel crosstalk and direct excitation of the acceptor, it also excels in removal of autofluorescence or background contaminations and in correcting chromatic aberrations, often overlooked factors that severely undermine FRET experiments. Finally, SpRET quantitatively reports absolute rather than relative FRET efficiency values, as well as the acceptor-to-donor molar ratio, which is critical for full and proper interpretation of FRET experiments. Thus, SpRET serves as an advanced, improved, and powerful tool in the cell biologists toolbox.

CaRuby-Nano: a novel high affinity calcium probe for dual color imaging

The great demand for long-wavelength and high signal-to-noise Ca2+ indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments. DOI: http://dx.doi.org/10.7554/eLife.05808.001

Lighting up neural networks using a new generation of genetically encoded calcium sensors.

Two improved genetically encoded calcium indicators—based on structure-guided sensor design or on precise subcellular targeting to presynaptic boutons—allow single spikes to be detected in genetically defined populations of neurons and synapses in vivo.

Functionalizable red emitting calcium sensor bearing a 1,4-triazole chelating moiety

Herein we developed a functionalizable OFF-ON red emitting fluorescent calcium probe based on a new chelating system formed by CuAAC click chemistry (Huisgen cycloaddition). The pro-sensor 7 which is not sensitive to Ca2+, contains an alkyne moiety that, upon the click reaction, forms a chelating group involving the 1,4-triazole. Probe 10 exhibited good sensitivity towards calcium (K-d = 5.8 mu M) and zinc (5.6 mu M) with a high dynamic range (65 fold fluorescence increase), high quantum yield (0.59) and showed very low fluorescence enhancement in the presence of a high concentration of Mg2+. We extended this method and generated two dextran conjugates in order to compare their sensing properties with those of the molecular form of 10.

Twitching towards the ideal calcium sensor

A new family of genetically encoded ratiometric calcium indicators optimized for imaging of calcium signals in vivo exhibits near-linear fluorescence dynamics while minimizing artifacts caused by movement.

Paclitaxel-eluting balloon dilation of biliary anastomotic stricture after liver transplantation

AIM To investigate the safety and effectiveness of endoscopic therapy with a paclitaxel-eluting balloon (PEB) for biliary anastomotic stricture (AS) after liver transplantation (LT). METHODS This prospective pilot study enrolled 13 consecutive eligible patients treated for symptomatic AS after LT at the University Hospital of Münster between January 2011 and March 2014. The patients were treated by endoscopic therapy with a PEB and followed up every 8 wk by endoscopic retrograde cholangiopancreatography (ERCP). In cases of re-stenosis, further balloon dilation with a PEB was performed. Follow-up was continued until 24 mo after the last intervention. RESULTS Initial technical feasibility, defined as successful balloon dilation with a PEB during the initial ERCP procedure, was achieved in 100% of cases. Long-term clinical success (LTCS), defined as no need for further endoscopic intervention for at least 24 mo, was achieved in 12 of the 13 patients (92.3%). The mean number of endoscopic interventions required to achieve LTCS was only 1.7 ± 1.1. Treatment failure, defined as the need for definitive alternative treatment, occurred in only one patient, who developed recurrent stenosis with increasing bile duct dilatation that required stent placement. CONCLUSION Endoscopic therapy with a PEB is very effective for the treatment of AS after LT, and seems to significantly shorten the overall duration of endoscopic treatment by reducing the number of interventions needed to achieve LTCS.

Single-operator cholangioscopy for biliary complications in liver transplant recipients

AIM To evaluate cholangioscopy in addition to endoscopic retrograde cholangiopancreatography (ERCP) for management of biliary complications after liver transplantation (LT). METHODS Twenty-six LT recipients with duct-to-duct biliary reconstruction who underwent ERCP for suspected biliary complications between April and December 2016 at the university hospital of Muenster were consecutively enrolled in this observational study. After evaluating bile ducts using fluoroscopy, cholangioscopy using a modern digital single-operator cholangioscopy system (SpyGlass DS™) was performed during the same procedure with patients under conscious sedation. All patients received peri-interventional antibiotic prophylaxis and bile was collected during the intervention for microbial analysis and for antibiotic susceptibility testing. RESULTS Thirty-three biliary complications were found in a total of 22 patients, whereas four patients showed normal bile ducts. Anastomotic strictures were evident in 14 (53.8%) patients, non-anastomotic strictures in seven (26.9%), biliary cast in three (11.5%), and stones in six (23.1%). A benefit of cholangioscopy was seen in 12 (46.2%) patients. In four of them, cholangioscopy was crucial for selective guidewire placement prior to planned intervention. In six patients, biliary cast and/or stones failed to be diagnosed by ERCP and were only detectable through cholangioscopy. In one case, a bile duct ulcer due to fungal infection was diagnosed by cholangioscopy. In another case, signs of bile duct inflammation caused by acute cholangitis were evident. One patient developed post-interventional cholangitis. No further procedure-related complications occurred. Thirty-seven isolates were found in bile. Sixteen of these were gram-positive (43.2%), 12 (32.4%) were gram-negative bacteria, and Candida species accounted for 24.3% of all isolated microorganisms. Interestingly, only 48.6% of specimens were sensitive to prophylactic antibiotics. CONCLUSION Single-operator cholangioscopy can provide important diagnostic information, helping endoscopists to plan and perform interventional procedures in LT-related biliary complications.