Manipulation Steps Research Articles

Enhancing droplet transition capabilities using sloped microfluidic channel geometry for stable droplet operation.

Droplet-based microfluidics technology allows for the generation and control of droplets that function as independent chemical and biological reactors, enabling broad ranges of high-throughput assays. As more complex multi-step assays are being realized in droplet format, maintaining droplet stability throughout the assay becomes a critical requirement. Unfortunately, as droplets go through multiple manipulation steps, droplet breakage is commonly seen, especially where droplets have to go through sharp transitions in direction and shape. Standard microfabrication techniques typically result in inherent sharp geometry in Z-direction due to their two-dimensional fabrication nature. Recent advancement in micro- and nano- fabrication technology using two-photon polymerization (2PP) is enabling complex 3D microstructures with sub-micrometer resolution to be readily fabricated. Here, utilizing this microfabrication technique, we present a simple solution to the droplet stability challenge by utilizing sloped-geometry microfluidic channels to enable microdroplets to smoothly transition between microfluidic channels having two different heights without breakage. The technique and innovation demonstrated here have the potential to replace conventional droplet microfluidic device fabrication approaches and enable droplet microfluidic platforms to achieve significantly higher level of efficiency, accuracy, and stability never realized before.

The transcriptome of Pinus pinaster under Fusarium circinatum challenge

BackgroundFusarium circinatum, the causal agent of pitch canker disease, poses a serious threat to several Pinus species affecting plantations and nurseries. Although Pinus pinaster has shown moderate resistance to F. circinatum, the molecular mechanisms of defense in this host are still unknown. Phytohormones produced by the plant and by the pathogen are known to play a crucial role in determining the outcome of plant-pathogen interactions. Therefore, the aim of this study was to determine the role of phytohormones in F. circinatum virulence, that compromise host resistance.ResultsA high quality P. pinaster de novo transcriptome assembly was generated, represented by 24,375 sequences from which 17,593 were full length genes, and utilized to determine the expression profiles of both organisms during the infection process at 3, 5 and 10 days post-inoculation using a dual RNA-sequencing approach. The moderate resistance shown by Pinus pinaster at the early time points may be explained by the expression profiles pertaining to early recognition of the pathogen, the induction of pathogenesis-related proteins and the activation of complex phytohormone signaling pathways that involves crosstalk between salicylic acid, jasmonic acid, ethylene and possibly auxins. Moreover, the expression of F. circinatum genes related to hormone biosynthesis suggests manipulation of the host phytohormone balance to its own benefit.ConclusionsWe hypothesize three key steps of host manipulation: perturbing ethylene homeostasis by fungal expression of genes related to ethylene biosynthesis, blocking jasmonic acid signaling by coronatine insensitive 1 (COI1) suppression, and preventing salicylic acid biosynthesis from the chorismate pathway by the synthesis of isochorismatase family hydrolase (ICSH) genes. These results warrant further testing in F. circinatum mutants to confirm the mechanism behind perturbing host phytohormone homeostasis.

Fast Small-Scale Membrane Protein Purification and Grid Preparation for Single-Particle Electron Microscopy.

The ongoing development of single-particle cryo-electron microscopy (cryo-EM) is leading to fast data acquisition, data processing, and protein structure elucidation. Quick and reliable methods to go from protein purification and optimization to grid preparation will significantly improve the reach and power of cryo-EM. Such methods would particularly constitute a tremendous advantage in structural biology of membrane proteins, whose published structures stay still far behind the number of soluble protein structures. Here we describe a fast, low-cost, and user-friendly method for the purification and cryo-EM analysis of a recombinant membrane protein. This method minimizes the amount of starting material and manipulation steps needed to go from purification to grid preparation, and could potentially be expanded to other membrane protein purification systems for its direct application in structure determination by single-particle cryo-EM.

Clinical validation of the Lumipulse G cerebrospinal fluid assays for routine diagnosis of Alzheimer\u2019s disease

BackgroundOngoing efforts within the Alzheimer’s disease (AD) field have focused on improving the intra- and inter-laboratory variability for cerebrospinal fluid (CSF) biomarkers. Fully automated assays offer the possibility to eliminate sample manipulation steps and are expected to contribute to this improvement. Recently, fully automated chemiluminescence enzyme immunoassays for the quantification of all four AD biomarkers in CSF became available. The aims of this study were to (i) evaluate the analytical performance of the Lumipulse G β-Amyloid 1-42 (restandardized to Certified Reference Materials), β-Amyloid 1-40, total Tau, and pTau 181 assays on the fully automated LUMIPULSE G600II; (ii) compare CSF biomarker results of the Lumipulse G assays with the established manual ELISA assays (INNOTEST®) from the same company (Fujirebio); and (iii) establish cut-off values and the clinical performance of the Lumipulse G assays for AD diagnosis.MethodsIntra- and inter-assay variation was assessed in CSF samples with low, medium, and high concentrations of each parameter. Method comparison and clinical evaluation were performed on 40 neurological controls (NC) and 80 patients with a diagnosis of probable AD supported by a follow-up ≥ 3 years and/or positive amyloid PET imaging. A small validation cohort of 10 NC and 20 AD patients was also included to validate the cut-off values obtained on the training cohort.ResultsThe maximal observed intra-assay and inter-assay coefficients of variation (CVs) were 3.25% and 5.50%, respectively. Method comparisons revealed correlation coefficients ranging from 0.89 (for Aβ40) to 0.98 (for t-Tau), with those for Aβ42 (0.93) and p-Tau (0.94) in-between. ROC curve analysis showed area under the curve values consistently above 0.85 for individual biomarkers other than Aβ40, and with the Aβ42/40, Aβ42/t-Tau, and Aβ42/p-Tau ratios outperforming Aβ42. Validation of the cut-off values in the independent cohort showed a sensitivity ranging from 75 to 95% and a specificity of 100%. The overall percentage of agreement between Lumipulse and INNOTEST was very high (> 87.5%).ConclusionsThe Lumipulse G assays show a very good analytical performance that makes them well-suited for CSF clinical routine measurements. The good clinical concordance between the Lumipulse G and INNOTEST assays facilitates the implementation of the new method in routine practice.

Synthesis of Heparan Sulfate- and Dermatan Sulfate-Related Oligosaccharides via Iterative Chemoselective Glycosylation Exploiting Conformationally Disarmed [2.2.2] l-Iduronic Lactone Thioglycosides.

Heparan sulfate (HS) and dermatan sulfate (DS) are l-iduronic acid containing glycosaminoglycans (GAGs) which are implicated in a number of biological processes and conditions including cancer and viral infection. Chemical synthesis of HS and DS is required to generate structurally defined oligosaccharides for a biological study. Herein, we present a new synthetic approach to HS and DS oligosaccharides using chemoselective glycosylation which relies on a disarmed [2.2.2] l-ido lactone motif. The strategy provides a general approach for iterative-reducing end chain extension, using only shelf-stable thioglycoside building blocks, exploiting a conformational switch to control reactivity, and thus requires no anomeric manipulation steps between glycosylations.

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo

Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development.

A Device for Preloaded, Trifolded Grafts to Facilitate Descemet Membrane Endothelial Keratoplasty

Abstract Descemet membrane endothelial keratoplasty (DMEK) is the most efficacious partial endothelial keratoplasty, offering the highest likelihood of 20/20 vision and the shortest recovery times. However, current devices do not effectively address the difficult step of unrolling the corneal graft within the eye which is the greatest barrier to DMEK adoption. Here, we present the design, development, and testing of a novel device that simplifies and standardizes DMEK graft transplantation. The patent-pending cartridge facilitates the trifold technique, a graft preparation method that allows the graft to naturally unroll in the eye and reduces time-consuming manipulation steps required in alternative techniques. Injection molding was used to manufacture devices which were tested with research-grade corneal grafts. The cartridge's design allows it to fit into a typical corneoscleral incision, maintains trifolded grafts (p = 0.006), and reduces endothelial cell loss (ECL) (p = 0.049). These results demonstrate that the device introduced here is suitable for DMEK and may simplify this procedure for corneal surgeons.

Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes.

Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family Enterobacteriaceae, as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.

Optical Temperature Control Unit and Convolutional Neural Network for Colorimetric Detection of Loop-Mediated Isothermal Amplification on a Lab-On-A-Disc Platform.

Lab-on-a-disc (LOD) has emerged as a promising candidate for a point-of-care testing (POCT) device because it can effectively integrate complex fluid manipulation steps using multiple layers of polymeric substrates. However, it is still highly challenging to design and fabricate temperature measurement and heating system in non-contact with the surface of LOD, which is a prerequisite to successful realization of DNA amplification especially with a rotatable disc. This study presents a Lab-on-a-disc (LOD)-based automatic loop-mediated isothermal amplification (LAMP) system, where a thermochromic coating (<~420 µm) was used to distantly measure the chamber’s temperature and a micro graphite film was integrated into the chamber to remotely absorb laser beam with super high efficiency. We used a deep learning network to more consistently analyze the product of LAMP than we could with the naked eye. Consequently, both temperature heating and measurement were carried out without a physical contact with the surface of LOD. The experimental results show that the proposed approach, which no previous work has attempted, was highly effective in realizing LAMP in LOD.

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems.

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

Improve your Galaxy text life: The Query Tabular Tool.

Galaxy provides an accessible platform where multi-step data analysis workflows integrating disparate software can be run, even by researchers with limited programming expertise. Applications of such sophisticated workflows are many, including those which integrate software from different 'omic domains (e.g. genomics, proteomics, metabolomics). In these complex workflows, intermediate outputs are often generated as tabular text files, which must be transformed into customized formats which are compatible with the next software tools in the pipeline. Consequently, many text manipulation steps are added to an already complex workflow, overly complicating the process. In some cases, limitations to existing text manipulation are such that desired analyses can only be carried out using highly sophisticated processing steps beyond the reach of even advanced users and developers. For users with some SQL knowledge, these text operations could be combined into single, concise query on a relational database. As a solution, we have developed the Query Tabular Galaxy tool, which leverages a SQLite database generated from tabular input data. This database can be queried and manipulated to produce transformed and customized tabular outputs compatible with downstream processing steps. Regular expressions can also be utilized for even more sophisticated manipulations, such as find and replace and other filtering actions. Using several Galaxy-based multi-omic workflows as an example, we demonstrate how the Query Tabular tool dramatically streamlines and simplifies the creation of multi-step analyses, efficiently enabling complicated textual manipulations and processing. This tool should find broad utility for users of the Galaxy platform seeking to develop and use sophisticated workflows involving text manipulation on tabular outputs.

Improve your Galaxy text life: The Query Tabular Tool

Galaxy provides an accessible platform where multi-step data analysis workflows integrating disparate software can be run, even by researchers with limited programming expertise. Applications of such sophisticated workflows are many, including those which integrate software from different ‘omic domains (e.g. genomics, proteomics, metabolomics). In these complex workflows, intermediate outputs are often generated as tabular text files, which must be transformed into customized formats which are compatible with the next software tools in the pipeline. Consequently, many text manipulation steps are added to an already complex workflow, overly complicating the process. In some cases, limitations to existing text manipulation are such that desired analyses can only be carried out using highly sophisticated processing steps beyond the reach of even advanced users and developers. For users with some SQL knowledge, these text operations could be combined into single, concise query on a relational database. As a solution, we have developed the Query Tabular Galaxy tool, which leverages a SQLite database generated from tabular input data. This database can be queried and manipulated to produce transformed and customized tabular outputs compatible with downstream processing steps. Regular expressions can also be utilized for even more sophisticated manipulations, such as find and replace and other filtering actions. Using several Galaxy-based multi-omic workflows as an example, we demonstrate how the Query Tabular tool dramatically streamlines and simplifies the creation of multi-step analyses, efficiently enabling complicated textual manipulations and processing. This tool should find broad utility for users of the Galaxy platform seeking to develop and use sophisticated workflows involving text manipulation on tabular outputs.

Intracameral lidocaine as supplement to classic topical anesthesia for relieving ocular pain in cataract surgery.

To evaluate safety, efficacy, and patient adherence of intracameral lidocaine as supplement of classic topical anesthetic drops in cataract surgery. A prospective and controlled trial including a large cohort of 1650 individuals suffering with bilateral cataract not complicated, in program by phacoemulsification surgery, were randomly assigned to 2 different groups for the type of anesthesia received, 0.4% oxybuprocaine hydrochloride (INN) drops, and INN drops associated to intracameral 1% lidocaine hydrochloride monohydrate. At the end of surgery, tables were assigned to each patient indicating the degree of pain (0-3) felt during the operation. Thirty-two percent of patients in group 1 declared to have not felt any pain against the 77% of patients in group 2. Fifty-nine percent of patients in group 1 complained about only a slight discomfort against 20% of group 2 patients. Only a small percentage of patients in group 1 (5%) admitted severe pain, while no patient in group 2 admitted severe pain. Four patients of group 2 reported an episode of transient amaurosis, lasting several hours after surgery. Intracameral administration of lidocaine is a simple and secure method able to increase the analgesia during the cataract surgery, eliminating the discomfort and increasing also the cooperation of the patients during the steps of manipulation.

Tailored Gradient Morphologies and Anisotropic Surface Patterns in Polymer Blends

AbstractGradient morphologies and surface patterns can provide a template for smart materials with tailored local properties. Fabricating such structures in a one‐step melt‐extrusion process will be most beneficial as they usually require several manipulation steps. In this study, gradient morphologies are developed by melt‐spinning matrix‐dispersed polypropylene/polystyrene blends as a template system and dynamic control of their formation is achieved, which further leads to anisotropic surface patterns. Cross and longitudinal sections and the surface of extruded fibers are evaluated by scanning electron microscopy. Distinct microstructure gradients regarding size and shape of the dispersed phase are formed in the bulk. After dissolving the dispersed phase, ordered (sub)micron channels appear on the surface, which are further refined by hot‐drawing. The resulting textures form anisotropic grooves along the fiber axis on the surface, as verified by atomic force microscopy. Capillary theory is applied to elucidate the formation mechanisms of gradient microstructures in polymer blends during processing. It is shown that all these features can be tailor‐made by using the extrusion die as a design tool. By transferring the knowledge to suitable polymer blends and processes, the designed gradient and surface structures can find applications, for instance, in materials with adaptive mechanical or interfacial properties.

Improve your Galaxy text life: The Query Tabular Tool

Galaxy provides an accessible platform where multi-step data analysis workflows integrating disparate software can be run, even by researchers with limited programming expertise. Applications of such sophisticated workflows are many, including those which integrate software from different ‘omic domains (e.g. genomics, proteomics, metabolomics). In these complex workflows, intermediate outputs are often generated as tabular text files, which must be transformed into customized formats which are compatible with the next software tools in the pipeline. Consequently, many text manipulation steps are added to an already complex workflow, overly complicating the process and decreasing usability, especially for non-expert bench researchers focused on obtaining results. In some cases, limitations to existing text manipulation are such that desired analyses can only be carried out using highly sophisticated processing steps beyond the reach of most users. As a solution, we have developed the Query Tabular Galaxy tool, which leverages a SQLite database generated from tabular input data. This database can be queried and manipulated to produce transformed and customized tabular outputs compatible with downstream processing steps. Regular expressions can also be utilized for even more sophisticated manipulations, such as find and replace and other filtering actions. Using several Galaxy-based multi-omic workflows as an example, we demonstrate how the Query Tabular tool dramatically streamlines and simplifies the creation of multi-step analyses, efficiently enabling complicated textual manipulations and processing. This tool should find broad utility for users of the Galaxy platform seeking to develop and use sophisticated workflows involving text manipulation on tabular outputs.

Smart spectrophotometric methods based on normalized spectra for simultaneous determination of alogliptin and metformin in their combined tablets

Alogliptin (ALO) and metformin (MET) are coformulated for the treatment of type II diabetes mellitus. ALO is estimated at its λmax 277 nm (0D), while MET was determined accurately by four spectrophotometric methods with minimum manipulation steps based on normalized division spectrum namely; ratio difference, advanced amplitude modulation, first derivative ratio (1DD) and mean centering of the ratio spectra spectrophotometric methods. Linearity was acceptable over the concentration ranges of 5–40 and 2–16 μg/mL for ALO and MET, respectively. Accuracy and precision of the suggested methods were found to be within the acceptable limit. The specificity was inspected by analyzing laboratory prepared mixtures of the above drugs and their pharmaceutical preparation. The results of proposed and reported methods were statistically compared showing no significant difference regarding accuracy and precision. The developed methods could be applied for routine analysis of the cited drugs in quality control laboratories.

Spectrophotometric resolution of the severely overlapped spectra of clotrimazole with dexamethasone in cream dosage form by mathematical manipulation steps

Several spectrophotometric techniques were recently conducted for the determination of binary mixtures of clotrimazole (CLT) and dexamethasone acetate (DA) without any separation procedure. The methods were based on generation of ratio spectra of mixture then applying simple mathematic manipulation. The zero order absorption spectra of both drugs could be obtained by the constant center (CC) method. The concentration of both CLT and DA could be obtained by constant value via amplitude difference (CV-AD) method depending on ratio spectra, Ratio difference (RD) method where the difference between the amplitudes at two wavelengths (ΔP) on the ratio spectra could eliminate the contribution of the interfering substance and bring the concentration of the other, and the derivative ratio (DD1) method where the derivative of the ratio spectra was able to determine the drug of interest without any interference of the other one. While the concentration of DA could be measured after graphical manipulation as concentration using the novel advanced concentration value method (ACV). Calibration graphs were linear in the range of 75–550 μg/mL for CLT and 2–20 μg/mL for DA. The methods applied to the binary mixture under study were successfully applied for the simultaneous determination of the two drugs in synthetic mixtures and in their combined form Mycuten-D cream. The results obtained were compared statistically to each other and to the official methods.

The many faces of data-centric workflow optimization: a survey

Workflow technology is rapidly evolving and, rather than being limited to modeling the control flow in business processes, is becoming a key mechanism to perform advanced data management, such as big data analytics. This survey focuses on data-centric workflows (or workflows for data analytics or data flows), where a key aspect is data passing through and getting manipulated by a sequence of steps. The large volume and variety of data, the complexity of operations performed, and the long time such workflows take to compute give rise to the need for optimization. In general, data-centric workflow optimization is a technology in evolution. This survey focuses on techniques applicable to workflows comprising arbitrary types of data manipulation steps and semantic inter-dependencies between such steps. Further, it serves a twofold purpose: firstly, to present the main dimensions of the relevant optimization problems and the types of optimizations that occur before flow execution and secondly, to provide a concise overview of the existing approaches with a view to highlighting key observations and areas deserving more attention from the community.

Laser-Capture Microdissection for the Analysis of Rat and Human Spinal Cord Ependyma by qPCR.

In the last few decades many efforts have been dedicated to decipher the nature and regenerative potential of neurogenic niches and endogenous stem cells after damage of the central nervous system. In the spinal cord, it has been largely focused on the ependymal region, which hosts neural precursors/stem cells (NSC) in rodents but differs between species and ages. In the current chapter, we detail our protocol to study the gene expression profile of this region using fresh frozen blocks of rat and human post-mortem spinal cords. We describe how to prepare and process those tissues, how to identify and dissect the ependymal region using Laser-Capture Microdissection (LCMD), and how to isolate and amplify RNA with different integrity states to finally obtain enough material for performing gene expression assays using Taqman® Low Density Arrays. LCMD technique maintains tissue integrity allowing for subsequent analysis without manipulation steps that may alter molecular properties of cells and the eventual loss of delicate cell types in comparison with other approaches that require previous disaggregation of the tissue and cell manipulation before isolation.

Experimental Simulation of Clinical Borderline Situations in Temporal Bone Specimens After Ossiculoplasty.

One reason for insufficient hearing improvement with a distinct air-bone gap after ossiculoplasty with implantation of partial or total ossicular replacement prostheses can be the dislocation or minimal shifting of the prosthesis. The aim of this study was the simulation of common clinical borderline situations with minimal shifting of the prosthesis in temporal bone specimens after ossiculoplasty. It was furthermore the goal to identify these specific situations through imaging by cone beam computed tomography (cbCT) and direct visual inspection using the operation microscope. Additionally, the functional status was evaluated using laser-Doppler vibrometry (LDV). We used a total of four temporal bone specimens for this study. A reconstruction with a partial ossicular replacement prostheses was performed in three specimens and with a total ossicular replacement prostheses in one specimen, with good initial acoustic properties. Subsequently, one specific type of prosthesis failure was simulated in each specimen, respectively, by minimally shifting, tilting, or bending the prostheses from their initial positions. These changes were introduced step-by-step until a borderline situation just short of complete acoustic decoupling was reached. Each step was examined using both LDV and cbCT and observed through the operation microscope. LDV was able to quantify the mechanic function of the ossicular chain after most of the manipulation steps by demonstrating the effect of any shifting of the prosthesis on the middle ear transfer function. However, in some situations, the middle ear transfer function was better with a visually more advanced failure of the prosthesis. In addition, cbCT showed most of the steps with excellent resolution and was able to delineate changes in soft tissue (e.g., cartilage covering). cbCT seems to be a promising imaging technique for middle ear problems. As cbCT and LDV exhibited slightly different advantages and disadvantages regarding the demonstration of borderline situations, the combination of both techniques allowed for a more precise evaluation of middle ear reconstructions. Knowledge of the specific characteristics of these methods and their possible combination might help otologists and otosurgeons to refine indications for revision surgery and improve their personal patient counseling.