Filament Dimensions Research Articles

Coaxial nozzle-assisted electrohydrodynamic printing for microscale 3D cell-laden constructs.

Cell printing has found wide applications in biomedical fields due to its unique capability in fabricating living tissue constructs with precise control over cell arrangements. However, it is still challenging to print cell-laden 3D structures simultaneously with high resolution and high cell viability. Here a coaxial nozzle-assisted electrohydrodynamic cell printing strategy was developed to fabricate living 3D cell-laden constructs. Critical process parameters such as feeding rate and stage moving speed were evaluated to achieve smaller hydrogel filaments. The effect of CaCl2 feeding rate on the printing of 3D alginate hydrogel constructs was also investigated. The results indicated that the presented strategy can print 3D hydrogel structures with relatively uniform filament dimension (about 80 μm) and cell distribution. The viability of the encapsulated cells was over 90%. We envision that the coaxial nozzle-assisted electrohydrodynamic printing will become a promising cell printing strategy to advance biomedical innovations.

Morphological and Anatomical Study to The Colchicum szovitsii from Colchicaceae Family in Iraq

Background: Colchicum szovitsii it is a wild perennial herbaceous plant grows in Iraq, it is medical and poisonous herb contain colchicine (alkaloid have cytotoxic effects) especially in corm and seeds. All parts are highly toxic if eaten. Handling of corms may cause skin allergy. Methods: This investigation present morphological and anatomical features of this plant for the first time in Iraq. (3) Results: The corm shape oblong to oblong-ovoid, the shape of the leaf is linear to oblong-lanceolate, undifferentiated perianth with 6 white oblanceolate tepals, the stamens epiphyllous. The anatomical study observed isolateral leaf, uniseriate epidermis and anomocytic stomata, and the mesophyll consisting many layers of irregular spongy paranchyma (chloranchyma), the type of vascular bundle is collateral. Conclusions: In this study we found that C. szovitsii distributed in Iraq have different morphological and anatomical features from the same species in Turkey and Iran like the dimension of corms, leaves, stamens, filament, anthers and capsule.

Advancing scaffold porosity through a machine learning framework in extrusion based 3D bioprinting

Three Dimensional (3D) bioprinting holds great promise for tissue and organ regeneration due to its inherent capability to deposit biocompatible materials containing live cells in precise locations. Extrusion-based 3D bioprinting (EBP) method stands out for its ability to achieve a higher cell release rate, ensuring both external and internal scaffold structures. The systematic adjustment of key process parameters of EBP, including nozzle diameter, printing speed, print distance, extrusion pressure, material fraction, and viscosity allows for precise control over filament dimensions, ultimately shaping the desired scaffold porosity as per user specifications. However, managing these factors with all possible interactions simultaneously to achieve the desired filament width can be intricate and resource intensive. This study presents a novel framework designed to construct a predictive model for the filament width of 3D bioprinted scaffolds for various process parameters. A total of 157 experiments have been conducted under various combinations of process parameters and biomaterial’s weight fraction for this study purpose. A regression-based machine learning approach is employed to develop the predictive model utilizing Adj. R2, Mallow’s Cp, and Bayesian Information Criterion (BIC). Following model development, rigorous experimental validations are conducted to assess the accuracy and reliability of the model. Based on the cross-validation of randomly split test data, Adj. R2 model emerges as the highest performing machine learning model (Mean Squared Error, MSE = 0.0816) compared to Mallow’s Cp and BIC (MSE = 0.0841 and 0.0877, respectively) models. The comparative analysis results between the experimental and model’s data demonstrate that our predictive model achieves an accuracy of approximately 85% in filament width prediction. This framework presents a significant advancement in the precise control and optimization of 3D bioprinted scaffold fabrication, offering valuable insights for the advancement of tissue engineering and regenerative medicine applications.

Towards a predictive understanding of direct ink writing of graphene-based inks

Direct ink writing (DIW) presents a flexible and resource-efficient approach towards the prototyping of functional materials and devices with complex shapes. Printed functional materials for electronic devices depend on conductive fillers such as graphene nanoplatelets (GNPs), which are increasingly popular in printed electronics and energy materials thanks to their low cost, non-toxicity and high specific surface area. However, non-spherical colloids with large filler-to-nozzle size ratios like GNPs present a challenge for high-resolution DIW due to risk of nozzle clogging. As DIW of platelet-based inks is gaining traction in several fields, the feasibility of high-resolution DIW of platelet-based inks is demonstrated here on the example of GNPs (< 50 μm). A workflow for the combined optimization of ink rheology and printing process parameters was developed to gain a predictive understanding of filament quality and morphology. Using two inks and two nozzle diameters per ink, filaments ranging from <100 – 1200 μm in width and 30 – 300 μm in height were produced, with conductivities suitable for application in sensors or electrodes. The derived predictive models were successfully deployed to predict filament dimensions and to achieve excellent print quality even for fine sub-nozzle size structures with very high filler-to-nozzle size ratios within only one iteration of the workflow. With this study, we advocate for the integrated development of materials for processes and processes for materials. This study will benefit high-resolution rapid prototyping of a large class of functional materials for wearable electronics, sensors, RF passives, energy materials and tissue engineering.

Material Extrusion Filament Width and Height Prediction via Design of Experiment and Machine Learning.

The dimensions of material extrusion 3D printing filaments play a pivotal role in determining processing resolution and efficiency and are influenced by processing parameters. This study focuses on four key process parameters, namely, nozzle diameter, nondimensional nozzle height, extrusion pressure, and printing speed. The design of experiment was carried out to determine the impact of various factors and interaction effects on filament width and height through variance analysis. Five machine learning models (support vector regression, backpropagation neural network, decision tree, random forest, and K-nearest neighbor) were built to predict the geometric dimension of filaments. The models exhibited good predictive performance. The coefficients of determination of the backpropagation neural network model for predicting line width and line height were 0.9025 and 0.9604, respectively. The effect of various process parameters on the geometric morphology based on the established prediction model was also studied. The order of influence on line width and height, ranked from highest to lowest, was as follows: nozzle diameter, printing speed, extrusion pressure, and nondimensional nozzle height. Different nondimensional nozzle height settings may cause the extruded material to be stretched or squeezed. The material being in a stretched state leads to a thin filament, and the regularity of processing parameters on the geometric size is not strong. Meanwhile, the nozzle diameter exhibits a significant impact on dimensions when the material is in a squeezing state. Thus, this study can be used to predict the size of printing filament structures, guide the selection of printing parameters, and determine the size of 3D printing layers.

Emission characteristics of bulk aerosols excited by externally focused femtosecond filaments.

The bulk aerosol emissions excited by externally focused femtosecond laser filaments are characterized using time-resolved plasma imaging and spectroscopy. Images of N2 and N2+ plasma fluorescence are used to characterize the filament dimensions. Emission profiles from bulk Sr aerosols are studied, showing that several localized emission regions in the filament begin to develop for lower repetition rates and higher pulse energies. Plasma temperature and electron density profiles are determined using particle emissions along the length of short- and long-focused filaments, and results are compared for on-axis and side-collected spectra. The use of on-axis collection enables the sampling of light emitted over the entire length of the filament; however, the necessary back-propagation of light makes on-axis collection susceptible to self-absorption as the optical path is extended through the filament plasma column formed in bulk aerosols.

Extrusion nozzle design and print parameter selections for 3D concrete printing

3D concrete printing is a rapidly emerging technology. This study presents theoretical formulations for concrete extrusion resistance as a function of nozzle parameters. Results show that the extrusion resistances are related to the extrusion velocity of concrete and the cone angle and length of the nozzle. Lowering the extrusion velocity and nozzle length can reduce the extrusion resistance in the nozzle. Based on the results, a nozzle was customized to investigate the effects of print parameters, namely, printing speed (50–200 mm/s), printing height (5–40 mm), and extrusion velocity (96–288 mm/s), on printing quality. The reduction of printing speed increased the limits of layer thickness. Finally, the relationship among nozzle dimensions, print parameters, and filament dimensions was found, which can be used to pre-estimate the printing quality before printing and help preset appropriate printing parameters.

Osteoconductivity of bone substitutes with filament-based microarchitectures: Influence of directionality, filament dimension, and distance.

63Additive manufacturing can be applied to produce personalized bone substitutes. At present, the major three-dimensional (3D) printing methodology relies on filament extrusion. In bioprinting, the extruded filament consists mainly of hydrogels, in which growth factors and cells are embedded. In this study, we used a lithography-based 3D printing methodology to mimic filament-based microarchitectures by varying the filament dimension and the distance between the filaments. In the first set of scaffolds, all filaments were aligned toward bone ingrowth direction. In a second set of scaffolds, which were derived from the identical microarchitecture but tilted by 90°, only 50% of the filaments were in line with the bone ingrowth direction. Testing of all tricalcium phosphate-based constructs for osteoconduction and bone regeneration was performed in a rabbit calvarial defect model. The results revealed that if all filaments are in line with the direction of bone ingrowth, filament size and distance (0.40-1.25 mm) had no significant influence on defect bridging. However, with 50% of filaments aligned, osteoconductivity declined significantly with an increase in filament dimension and distance. Therefore, for filament-based 3D- or bio-printed bone substitutes, the distance between the filaments should be 0.40 to 0.50 mm irrespective of the direction of bone ingrowth or up to 0.83 mm if perfectly aligned to it.

(Excellent Student Presentation Award) Confined Conducting Filaments in Resistive Random Access Memory By Al2O3 Nanodome-Shaped Arrays (NDSAs) Via Glancing-Angle Deposition

Resistive random access memory (RRAM) is vital to neuromorphic computing applications. However, filamentary RRAM cells are affected by transitions from abrupt switching to analog switching. In this study, we develop Al2O3 nanodome shaped arrays (NDSAs) by glancing angle deposition technology (GLAD) to geometrically confine the conducting filaments (CFs), for which conducting atomic force microscopy (C-AFM) was performed to analyze positions and dimensions of filaments. For the Pt/HfO2/75 % Al2O3 NDSAs/TiN device, the dimension of the CFs can be restricted to 10-12 nm, whereas for the Pt/HfO2/TiN device, the CFs were formed with the dimension of ~50 nm. The device first yielded multiple weak CFs that subsequently transformed into stronger and larger CFs when the coverage of Al2O3 NDSAs was reduced to 55 % while Pt/HfO2/75 % Al2O3 NDSAs/TiN device exhibited synaptic features with more linear potentiation and depression, demonstrating the analog switching. The controllable coverages of Al2O3 NDSAs render the geometric design more promising as a memristor for future applications in neuromorphic computing.

Additive manufacturing of complexly shaped SiC with high density via extrusion-based technique – Effects of slurry thixotropic behavior and 3D printing parameters

Additive manufacturing of dense SiC parts was achieved via an extrusion-based process followed by electrical-field assisted pressure-less sintering. The aim of this research was to study the effect of the rheological behavior of SiC slurry on the printing process and quality, as well as the influence of 3D printing parameters on the dimensions of the extruded filament, which are directly related to the printing precision and quality. Different solid contents and dispersant- Darvan 821A concentrations were studied to optimize the viscosity, thixotropy and sedimentation rate of the slurry. The optimal slurry was composed of 77.5 wt% SiC, Y2O3 and Al2O3 powders, 0.25 wt% dispersant and 0.01 wt% defoamer. The printing parameters studied included extrusion pressure, nozzle size, layer height and printing speed; the one that had the most prominent effect on filament width and height was indicated as layer height. The nozzle inner diameter of 1.04 mm, speed of 350 mm/min, layer height of 0.7 mm and extrusion air pressure of 0.31 MPa were the optimal printing parameters. Furthermore, the relationship between the printing parameters and the filament dimensions was successfully predicted by using machine learning and grey system theory. Finally, the relative density of the printed SiC parts sintered at 1900 oC reached 94.7±1.5%.

Extrusion 3D bioprinting of functional self-supporting neural constructs using a photoclickable gelatin bioink

Many in vitro models of neural physiology utilize neuronal networks established on two-dimensional substrates. Despite the simplicity of these 2D neuronal networks, substrate stiffness may influence cell morphology, network interactions and how neurons communicate and function. With this perspective, three-dimensional (3D) gel encapsulation is a powerful to recapitulating aspects of in vivo features, yet such an approach is often limited in terms of the level of resolution and feature size relevant for modelling aspects of brain architecture. Here, we report 3D bioplotting of rat primary cortical neural cells using a hydrogel system comprising gelatin norbornene (GelNB) and poly (ethylene glycol) dithiol (PEGdiSH). This bioink benefits from a rapid photo-click chemistry, yielding eight-layer crosshatch neural scaffolds and a filament width of 350 µm. The printability of this system depends on hydrogel concentration, printing temperature, extrusion pressure and speed. These parameters were studied via quantitative comparison between rheology and filament dimensions to determine the optimal printing conditions. Under optimal conditions, cell viability of bioprinted primary cortical neurons at day 1 (68 ± 2%) and at day 7 (68 ± 1%) were comparable to the 2D control group (72 ± 7%). The present study relates material rheology and filament dimensions to generate compliant free-standing neural constructs through bioplotting of low-concentration GelNB-PEGdiSH, which may provide a step forward to study 3D neuronal function and network formation.

Reduced Stochastic Resistive Switching in Organic‐Inorganic Hybrid Memristors by Vapor‐Phase Infiltration

AbstractResistive random‐access memory (RRAM) is promising for next‐generation data storage and non‐von Neumann computing hardware. However, tuning device switching characteristics and particularly, controlling their stochastic variation remain as critical challenges. Here, new organic‐inorganic hybrid RRAM media are reported whose bipolar switching characteristics and stochasticity can be controlled by vapor‐phase infiltration (VPI), an ex situ hybridization technique derived from atomic layer deposition. Hybrid RRAMs based on AlOx‐infiltrated SU‐8 feature facile tunability of device switching voltages, off‐state current, and on‐off ratio by adjusting the amount of infiltrated AlOx in the hybrid. Furthermore, a significant reduction in the stochastic, cycle‐to‐cycle variations of switching parameters is enabled by AlOx infiltration, driven by the infiltration‐induced changes in mechanical, dielectric, and chemical properties of organic medium and their influence on the dimension and formation characteristics of conductive filaments. Finally, multi‐level analog switching potentially useful for neuromorphic applications are demonstrated, along with direct, one‐step device patterning exploiting the negative‐tone resist feature of SU‐8. With the demonstrated control over switching characteristics and stochastic variation, combined with analog switching and one‐step patterning capabilities, the results not only present a novel hybrid medium for RRAM applications but also showcase the utility of VPI for developing new, high‐performance hybrid RRAM devices.

Morphological and botanical profile of Lachenalia cultivars

Constant selection of common ornamental geophytes increases activity in the field of traditional crop improvement by breeding and commercialisation of new cultivars. In this paper, the morphological and botanical attributes (inflorescence stem height, inflorescence length, stem diameter, flower number, pedicel length and distance, fullness ratio, inflorescence type, leaf characteristics, perianth shape and colour and filament orientation as well as tepal, filament, anther and ovary dimensions) as well as growth and flowering patterns (number of days from potting date to the beginning of flowering) of seven Lachenalia cultivars (‘Aqua Lady’, ‘Josephine’, ‘Radient’, ‘Rainbow Bells’, ‘Riana’, ‘Romaud’ and ‘Ronina’) possessing high ornamental value were described in detail. The obtained results indicate differences of the studied cultivars both in terms of the dimensions of individual plant elements, as well as in the colours of flowers and flowering time. More compact cultivars, as ‘Josephine’, ‘Radient’, ‘Riana’ and ‘Romaud’ are recommended for potted cultivation. ‘Rainbow Bells’, ‘Aqua Lady’ and ‘Ronina’ should rather be considered as cut flowers. Principal components analysis identified four principal components that accounted for 83.4% of the total variability.

Process capability analysis for fabrication of high elongated PVC polymer using extrusion process

Poly-vinyl chloride (PVC) is one of the most demandable, usable commonly used thermoplastic polymer. Lots of research work related to PVC polymer has been highlighted in the past work due to its better mechanical strength such as elongation, flexibility and dimensional accuracy. In the past few years, it has been trying to add some properties which helps PVC polymer are easily used in the 3D printing technology. In the present study main highlight is to make accurate dimension of feedstock filament using twin-screw extruder (TSE). In this research work an effort has been made to fabricate PVC based feedstock filament which helps 3D printing are to be performed with easy flexibility. In this research work graphite fine powder has been reinforced in PVC thermoplastic extrusion using TSE process. Variation of graphite fine powder is varied from 5% to 15% with the gap of 5. For this research work, Taguchi L9 (3^3) orthogonal array has been used for fabrication of 3D printer feedstock filaments. Process capability analysis has been performed on fabricated feed stock filament for commercially fabrications. In this research work dimensional accuracy has been determined.

Photonic lanterns: a practical guide to filament tapering

We present a detailed method of tapering and drawing photonic lanterns using a filament glass processing system. Single-mode fibers (SMFs) were stacked inside a low refractive index, fluorine-doped capillary, which was then heated and tapered to produce a transition from single-mode to multi-mode. Fabrication parameters were considered in four categories: method of preparation and stacking of SMFs into a capillary, heat and filament dimensions of the glass processor, capillary ID, and the use of vacuum during tapering. 19- and 37- fiber lanterns were drawn, demonstrating good fusion between SMF claddings, a clear differentiation between core and cladding in the multimode (MM) section, and well-ordered arrangements between SMFs, which is controlled during the tapering process. The transmission efficiency of a 19-fiber photonic lantern, compared to an MMF with the same core diameter and NA, has a relative transmission efficiency of 1.19 dB or 67.1%. The steps and parameters provided in this paper form a framework for fabricating quality photonic lanterns.

Dripping and jetting of semi-dilute polymer solutions co-flowing in co-axial capillaries

This work is focused on the mechanisms of the dripping and jetting flow modes of viscoelastic semi-dilute polyacrylamide aqueous solutions co-flowing with silicone oil in co-axial capillaries. A phase diagram of the dripping, jetting, and intermediate flow modes is established. It was found that in the dripping mode, the elongation velocity of the filament between the terminal droplet and the inner capillary is controlled solely by the continuous phase rate. At the same time, the decrease in the filament diameter is due to both stretching and outflow of the polymer solution into the terminal droplet. In the jetting mode, the thread diameter was found to evolve in three stages. In the first stage, the average jet velocity increases, whereas in the second and third stages, it becomes constant and corresponds to the velocity of the continuous phase. The transition from the second to the third stage is defined by the appearance of capillary waves resulting in the formation of the beads-on-string structure. In the third stage, the filament diameter between the neighbor beads decreases exponentially and is governed by the relaxation time, which strongly depends on polymer concentration, but does not depend on the continuous phase flow rate. A simple physical model was proposed for describing the evolution of dimensions of filaments and beads during development of jet capillary instability. The universal character of the evolution of filaments and beads sizes, which is independent of concentration of semi-diluted polymer solutions and flow rates of the continuous phase, is revealed.

Adaptive ordering and filament polymerization of cell cytoskeleton by tunable nanoarrays

Matrix adaptation reconstructs the architecture of cell, and has important implication in proper biological functioning. However, how the network of cytoskeleton filament undergoes reconstruction, reordering, and surface adaptation, requires a systematic investigation. Here, we show that surface sensing and adaptation occur correspondingly with related reorganization of cytoskeleton filaments (actin, tubulin, and vimentin). The microstructure of filament network is built by adaptive change of chemical polymerization on cytoskeleton filaments. The transition of cellular morphology, from spheroidal architecture on nanoarray to extending structure with stress fibers on flat surface, involves spatial reorganization and polymerization modulation of filaments. The dimension of filaments (diameter, orientation, and density) are changed accordingly to spatiotemporal distribution of cytoskeleton network. In addition, our findings elucidate how cell can tune their architecture at nanoscale by matrix adaptation, and provide a novel information on interplay between cytoskeleton and pathophysiology.

Preliminary study of in‐situ 3D bioprinted nano‐silicate biopolymer scaffolds for muscle repair in VML defects

IntroductionThe traumatic or surgical loss of skeletal muscle with resultant functional impairment (volumetric muscle loss (VML)) is a challenging clinical problem. The current standard treatment care for VML is autologous tissue grafts and physical therapy, which still faces the challenge of donor site morbidity in large defect cases. 3D printing techniques for tissue engineering allows the precise control of micropatterning by determining the dimensions of filaments, the size and shape of pores and the percentage of porosity of the scaffold. Our hypothesis is using 3D printing of biopolymer‐nanosilicate scaffolds in‐situ will enhance muscle regeneration in VML defects.Materials and MethodsMethacrylated gelatin (MAG) biopolymer Laponite (Lp) nano‐silicate scaffolds were prepared as a bio‐ink using a solution‐precipitation process. Our study includes designing and optimization of 3D printing parameters for printing MAG‐Lp scaffolds and in‐vitro evaluation of cell viability (live‐dead), proliferation, and differentiation of mouse myoblast precursor cells on these scaffolds. We further analyzed the in‐situ (directly in the defect) 3D printing (using a robocaster) in a rat VML model in tibialis anterior muscle to evaluate the feasibility, precision, flexibility and attachment of the scaffold to the defect.ResultsSilicon ion enhanced cell proliferation and differentiation (fusion index by 1.5 times and overexpression of myogenic factors) in mouse myoblast precursor cells as compared to control. Qualitative live/dead analysis showed more abundance of live myoblast precursor cells (green) and decreased number of dead cells (red) on MAG‐Lp surface as compared to MAG (Fig. 1). We also show successful printing abilities of MAG‐Lp scaffolds in the volumetric muscle loss defect (Fig. 2) along with increased attachment to the adjacent tissues. The animal also showed ability to stand on/flex the surgical limb, with sufficient grip strength to perform normal activity, immediately after recovering from the surgery.ConclusionOur preliminary findings conclude that 3D printing of MAG‐Lp can enhance myogenesis in myoblasts. We further conclude that successful proof of concept of an in‐situ bioprinting method of reconstructive scaffolds can be used for immediate repair and loading of muscles for VML injury treatment.Support or Funding InformationNIH/NIDCR (R03DE023872‐01, 1R56DE027964‐01A1‐01), Texas STARs award, Departmental Start‐up Funds, University of Texas at Arlington, College of Nursing and Health Innovation, Arlington, TXshowing live/dead assay on MAG and MAG‐Lp scaffolds. Green represents live cells, whereas red represents dead cells.Figure 1showing In‐situ 3D printing biopolymer scaffold in a rat volumetric muscle loss model in tibialis anterior muscle (20–25% muscle loss) along with UV/photo curingFigure 2

Single-particle measurements of filamentous influenza virions reveal damage induced by freezing.

Clinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantify and characterize the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to -70 °C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced their concentration, median length and the infectivity of the whole virion population. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by snap-freezing or incorporating the cryoprotectant DMSO. We recommend that functional studies of filaments characterize virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterizations of filaments that are required to understand their roles in natural influenza virus infections.

Single-particle measurements reveal damage to filamentous influenza virions during laboratory handling

Most laboratory strains of influenza virus produce near-spherical virions, but clinical isolates also produce extended filaments whose biophysical properties are understudied. Most functional studies of filamentous influenza viruses do not include data on the concentration or lengths of the virions, making it hard to interpret their sometimes contradictory results. Furthermore, anecdotal reports suggest that filaments are damaged during routine laboratory handling. Therefore, to understand filament function we require a tool to assess the number and dimensions of filaments in a sample and an assessment of how filaments respond to standard handling procedures. We initially sought to analyse filament populations using negative stain particle counting, but found that this was low-throughput and could not detect particles longer than 10 µm. Instead, we used confocal microscopy with semi-automated image analysis. This allowed a high-throughput, quantitative analysis of length distributions in filament populations. Using this, we assessed the effects of pipetting, vortexing, sonicating, clarification and freezing on filaments. Most procedures did not appreciably alter filament dimensions. Pipetting and vortexing both slightly reduced filament numbers, but their effects were only appreciable after extended treatment. In contrast, freezing substantially reduced the number and median length of filaments, as well as creating ‘kinks’ in filaments which suggest damage to the capsid. We conclude that confocal microscopy can provide the basic measurements needed to interpret functional studies of filamentous strains. Using this approach, we found that freezing filaments causes previously unappreciated damage, which should be considered when planning further research.