Comparison between the acoustic fundamental frequency of the voice and the vibration frequency of the vocal folds analyzed by digital kymography. / Comparação entre a frequência fundamental acústica da voz e a frequência de vibração das pregas vocais analisada pela videoquimografia digital.

PURPOSE: To compare the frequency of vocal fold opening variation, analyzed by digital kymography, with the fundamental voice frequency obtained by acoustic analysis, in individuals without laryngeal alteration. METHODS: Observational analytical cross-sectional study. The participants were forty-eight women and 38 men from 18 to 55 years of age. The evaluation was made by voice acoustic analysis, by the habitual emission of the vowel /a/ for 3 seconds, and days of the week, and digital kymography (DKG), by the habitual emission of the vowels /i/ and /ɛ/. The measurements analyzed were acoustic fundamental frequency (f0), extracted by the Computerized Speech Lab (CSL) program, and dominant frequency of the variation of right (R-freq) and left (L-freq) vocal fold opening, obtained through the KIPS image processing program. The mounting of the kymograms consisted in the manual demarcation of the region by vertical lines delimiting width and horizontal lines separating the posterior, middle and anterior thirds of the Rima glottidis. In the statistical analysis, the Anderson-Darling test was used to verify the normality of the sample. The ANOVA and Tukey tests were performed for the comparison of measurements between the groups. For the comparison of age between the groups, the Mann-Whitney test was used. RESULTS: There are no differences between the values of the frequency measurement analyzed by digital kymography, with the acoustic fundamental frequency, in individuals without laryngeal alteration. CONCLUSION: The values of the dominant frequency of the vocal folds opening variation, as assessed by digital kymography, and the acoustic fundamental frequency of the voice are similar, allowing comparison between these measurements in the multidimensional evaluation of the voice, in individuals without laryngeal alteration.

OBJETIVO: Comparar a frequência da variação da abertura das pregas vocais, analisada pela videoquimografia digital, com a frequência fundamental da voz, obtida através da análise acústica, em indivíduos sem alteração laríngea. MÉTODO: Trata-se de um estudo observacional analítico transversal. Participaram 48 mulheres e 38 homens, de 18 a 55 anos. A avaliação foi composta por análise acústica da voz, obtida pela emissão habitual da vogal /a/ durante 3 segundos, e os dias da semana, e pela videoquimografia digital (DKG), obtida pela emissão habitual das vogais /i/ e /ɛ/. As medidas analisadas foram a frequência fundamental acústica (f0), extraída pelo programa Computerized Speech Lab (CSL), e a frequência dominante da variação de abertura da prega vocal direita (D-freq) e esquerda (E-freq), obtidas através do programa de processamento de imagens KIPS. A montagem dos quimogramas constou na demarcação manual da região, compostas por linhas verticais que delimitaram largura da prega vocal e linhas horizontais que marcaram os terços posterior, médio e anterior da rima glótica. Na análise estatística, o teste Anderson-Darling foi utilizado para verificar a normalidade da amostra. Os testes ANOVA e Tukey foram realizados para a comparação das medidas entre os grupos. Para a comparação da idade entre os grupos, foi utilizado o teste Mann-Whitney. RESULTADOS: Não existem diferenças entre os valores da medida de frequência analisada pela videoquimografia digital, com a frequência fundamental acústica, em indivíduos sem alteração laríngea. CONCLUSÃO: Os valores da frequência dominante da variação de abertura das pregas vocais, avaliada pela videoquimografia digital, e a frequência fundamental acústica da voz são similares, permitindo uma comparação entre estas medidas na avaliação multidimensional da voz, em indivíduos sem alteração laríngea.

Evaluation of Voice and Vocal Fold Vibration after Thyroidectomy Using Two-Dimensional Scanning Digital Kymography and High-Speed Videolaryngoscopy.

OBJECTIVE: Vocal dysfunction is one of the major factors that affect the health-related quality of life of patients after thyroidectomy. Conventionally, voice changes after thyroidectomy have been evaluated by videostroboscopy and acoustic analysis. Recently, two-dimensional scanning digital kymography (2D DKG) and high-speed videolaryngoscopy (HSV) have been developed and have shown usefulness in accurately evaluating vocal fold vibration. This study aimed to evaluate changes of vocal fold vibration and voice after thyroidectomy using 2D DKG and HSV. MATERIALS AND METHODS: We evaluated the voice and vocal fold movement of 24 female patients who underwent thyroidectomy in a single tertiary hospital from December 2018 to October 2019. We obtained serial 2D DKG and HSV data one day before thyroidectomy, and 1 week and 1 month after surgery. We analyzed the peak glottal area of HSV, amplitude symmetry index, phase symmetry index, and open quotient using the 2D DKG data. The parameters were calculated at three levels of the vocal fold (line 1=anterior, line 2=middle, line 3=posterior). In the same period, we performed a voice analysis evaluating voice frequency, jitter, shimmer, and noise to harmonic ratio. We also assessed maximum phonation time and subjective voice changes with voice handicap index-10 questionnaires. RESULTS: Highest frequency (F-high), frequency range (F-range), and fundamental frequency (F0) decreased at 1 week and 1 month after thyroidectomy compared with preoperative values (P = 0.003, 0.004, <0.001 and P = 0.002, 0.015, 0.001 at 1 week and 1 month, respectively). The open quotient of 2D DKG in lines 1 and 2 increased at 1 week after thyroidectomy (P = 0.011, 0.006) and recovered to preoperative levels at 1 month postoperatively (P = 0.189, 0.153). Other quantitative measures by 2D DKG and HSV did not show significant changes between the preoperative and postoperative periods. In a correlation analysis between vocal parameters from the acoustic analysis and the values obtained from 2D DKG and HSV, significant negative correlations were observed between peak glottal area and three factors (F-high, F-range, and F0) at 1 month after surgery (r = -0.589, -0.529, -0.708; P = 0.002, 0.008, <0.001, respectively). There were positive correlations between phase symmetry indexes in lines 1 and 2 and shimmer at 1 week after thyroidectomy (r = 0.489, 0.425; P = 0.015, 0.038, respectively). Phase symmetry index in line 3 showed a significant negative correlation with maximum phonation time at both 1 week and 1 month after surgery (r = -0.497, -0.439; P = 0.013, 0.032, respectively). However, there was no correlation between total score on the voice handicap index-10 questionnaires and quantitative measurements of vocal fold vibration. CONCLUSION: 2D DKG and HSV may provide important information on vocal fold vibratory patterns after thyroidectomy, and measurements made with them were correlated with maximal phonation time and acoustic parameters such as F-high, F-range, F0, shimmer.

Comparative analysis of high-speed videolaryngoscopy images and sound data simultaneously acquired from rigid and flexible laryngoscope: a pilot study.

High-Speed Videoendoscopy (HSV) is becoming a robust tool for the assessment of vocal fold vibration in laboratory investigation and clinical practice. We describe the first successful application of flexible High Speed Videoendoscopy with innovative laser light source conducted in clinical settings. The acquired image and simultaneously recorded audio data are compared to the results obtained by means of a rigid endoscope. We demonstrated that the HSV recordings with fiber-optic laryngoscope have enabled obtaining consistently bright, color images suitable for parametrization of vocal fold oscillation similarly as in the case of the HSV data obtained from a rigid laryngoscope. The comparison of period and amplitude perturbation parameters calculated on the basis of image and audio data acquired from flexible and rigid HSV recording objectively confirm that flexible High-Speed Videoendoscopy is a more suitable method for examination of natural phonation. The HSV-based measures generated from this kymographic analysis are arguably a superior representation of the vocal fold vibrations than the acoustic analysis because their quantification is independent of the vocal tract influences. This experimental study has several implications for further research in the field of HSV application in clinical assessment of glottal pathologies nature and its effect on vocal folds vibrations.

Quantifying the polymerization dynamics of plant cortical microtubules using kymograph analysis.

The plant cortical microtubule array is a dynamic structure that confers cell shape and enables plants to alter their growth and development in response to internal and external cues. Cells use a variety of microtubule regulatory proteins to spatially and temporally modulate the intrinsic polymerization dynamics of cortical microtubules to arrange them into specific configurations and to reshape arrays to adapt to changing conditions. To obtain mechanistic insight into how particular microtubule regulatory proteins mediate the dynamic (re)structuring of cortical microtubule arrays, we need to measure their effect on the dynamics of cortical microtubules. In this chapter, we describe new ImageJ plugins to generate kymographs from time-lapse images and to analyze them to measure the parameters that quantitatively describe cortical microtubule dynamics.

Analyzing Intracellular Gradients in Pollen Tubes.

Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+ probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.

Kymolyzer, a Semi-Autonomous Kymography Tool to Analyze Intracellular Motility.

The movement of intracellular cargo, such as transcripts, proteins, and organelles, is fundamental to cellular function. Neurons, due to their long axons and dendrites, are particularly dependent on proper intracellular trafficking and vulnerable to defects in the movement of intracellular cargo that are noted in neurodegenerative and neurodevelopmental disorders. Accurate quantification of intracellular transport is therefore needed for studying the mechanisms of cargo trafficking, the influence of mutations, and the effects of potentially therapeutic pharmaceuticals. In this article, we introduce an algorithm called "Kymolyzer." The algorithm can quantify intracellular trafficking along a defined path, such as that formed by the aligned microtubules of axons and dendrites. Kymolyzer works as a semi-autonomous kymography software application. It constructs and analyzes kymographs to measure the movement and distribution of fluorescently tagged objects along a user-defined path. The algorithm can be used under a wide variety of experimental conditions and can extract a diverse array of motility parameters describing intracellular movement, including time spent in motion, percentage of objects in motion, percentage of objects that are stationary, and velocities of motile objects. This article serves as a user manual describing the design of Kymolyzer, providing a stepwise protocol for its use and illustrating its functions with common examples. © 2020 Wiley Periodicals LLC Basic Protocol: Kymolyzer, a semi-autonomous kymography tool to analyze intracellular motility.

BacStalk: A comprehensive and interactive image analysis software tool for bacterial cell biology.

Prokaryotic cells display a striking subcellular organization. Studies of the underlying mechanisms in different species have greatly enhanced our understanding of the morphological and physiological adaptation of bacteria to different environmental niches. The image analysis software tool BacStalk is designed to extract comprehensive quantitative information from the images of morphologically complex bacteria with stalks, flagella, or other appendages. The resulting data can be visualized in interactive demographs, kymographs, cell lineage plots, and scatter plots to enable fast and thorough data analysis and representation. Notably, BacStalk can generate demographs and kymographs that display fluorescence signals within the two-dimensional cellular outlines, to accurately represent their subcellular location. Beyond organisms with visible appendages, BacStalk is also suitable for established, non-stalked model organisms with common or uncommon cell shapes. BacStalk, therefore, contributes to the advancement of prokaryotic cell biology and physiology, as it widens the spectrum of easily accessible model organisms and enables highly intuitive and interactive data analysis and visualization.

Clinical Application of Two-Dimensional Scanning Digital Kymography in Discrimination of Diplophonia.

Purpose The purpose of this study was to investigate the characteristics of diplophonia using an auditory perception and multimodal simultaneous examination, which included sound waveform analysis, electroglottography (EGG), digital kymography (DKG), and 2-dimensional scanning digital kymography (2D DKG). Additionally, we compared the diagnostic accuracy of each method using a binary classifier in confusion matrix and convenience of discrimination, based on the time required for interpretation. Method One normophonic male, 12 patients with diplophonia, and 12 dysphonia patients without diplophonia were enrolled. A multimodal simultaneous evaluation was used to analyze the vibration pattern of diplophonia. Sensitivity, specificity, accuracy, area under the curve, and interpretation time were used to compare the various diagnostic methods. Discrimination was determined by 3 raters. Results There are 3 types of asymmetric vibratory patterns in diplophonia. The types are based on the oscillators vibrating at different frequencies: asymmetry of the left and right cords (6 subjects with unilateral palsy and 1 subject with vocal polyps), asymmetry of anterior and posterior cords (2 subjects with vocal polyps), and asymmetry of true and false cords (3 subjects with muscle tension dysphonia). All evaluation methods were useful as diagnostic tools, with all areas under the curve > .70. The diagnostic accuracy was highest with DKG (95.83%), followed by 2D DKG (83.33%), EGG (81.94%), auditory-perceptual evaluation (80.56%), and sound waveform (77.78%). The interpretation time was the shortest for auditory-perceptual evaluation (6.07 ± 1.34 s), followed by 2D DKG (10.04 ± 3.00 s), EGG (12.49 ± 2.76 s), and DKG (13.53 ± 2.60 s). Conclusions Auditory-perceptual judgment was the easiest and fastest method for experienced raters, but its diagnostic accuracy was lower than that of DKG or 2D DKG. The diagnostic accuracy of DKG was the highest, but 2D DKG allowed rapid interpretation and showed relatively high diagnostic accuracy, except in cases with space-occupying lesions. Supplemental Material https://doi.org/10.23641/asha.9911786.

KymoButler, a deep learning software for automated kymograph analysis.

Kymographs are graphical representations of spatial position over time, which are often used in biology to visualise the motion of fluorescent particles, molecules, vesicles, or organelles moving along a predictable path. Although in kymographs tracks of individual particles are qualitatively easily distinguished, their automated quantitative analysis is much more challenging. Kymographs often exhibit low signal-to-noise-ratios (SNRs), and available tools that automate their analysis usually require manual supervision. Here we developed KymoButler, a Deep Learning-based software to automatically track dynamic processes in kymographs. We demonstrate that KymoButler performs as well as expert manual data analysis on kymographs with complex particle trajectories from a variety of different biological systems. The software was packaged in a web-based 'one-click' application for use by the wider scientific community (http://kymobutler.deepmirror.ai). Our approach significantly speeds up data analysis, avoids unconscious bias, and represents another step towards the widespread adaptation of Machine Learning techniques in biological data analysis.

Many molecules and structures within cells have to move about to do their job. Studying these movements is important to understand many biological processes, including the development of the brain or the spread of viruses. Kymographs are images that represent the movement of particles in time and space. Unfortunately, tracing the lines that represent movement in kymographs of biological particles is hard to do automatically, so currently this analysis is done by hand. Manually annotating kymographs is tedious, time-consuming and prone to the researcher's unconscious bias. In an effort to simplify the analysis of kymographs, Jakobs et al. have developed KymoButler, a software tool that can do it automatically. KymoButler uses artificial intelligence to trace the lines in a kymograph and extract the information about particle movement. It speeds up analysis of kymographs by between 50 and 250 times, and comparisons show that it is as reliable as manual analysis. KymoButler is also significantly more effective than any previously existing automatic kymograph analysis programme. To make KymoButler accessible, Jakobs et al. have also created a website with a drag-and-drop facility that allows researchers to easily use the tool. KymoButler has been tested in many areas of biological research, from quantifying the movement of molecules in neurons to analysing the dynamics of the scaffolds that help cells keep their shape. This variety of applications showcases KymoButler's versatility, and its potential applications. Jakobs et al. are further contributing to the field of machine learning in biology with 'deepmirror.ai', an online hub with the goal of accelerating the adoption of artificial intelligence in biology.

Efficacy of Two-dimensional Scanning Digital Kymography in Evaluation of Atrophic Vocal Folds.

OBJECTIVE: The purpose of this study was to evaluate the clinical feasibility and diagnostic accuracy of two-dimensional scanning digital kymography (2D DKG) in patients with vocal cord atrophy before and after treatment. MATERIALS AND METHODS: We analyzed the characteristics of vocal fold vibration in five patients with unilateral vocal fold paralysis and five patients with presbyphonia. In patients with vocal cord paralysis, the status before and after intracordal injection was compared. Furthermore, in patients with presbyphonia, we compared the status before and after voice therapy (Seong-Tae Kim's laryngeal calibration technique). Quantitative parameters such as amplitude and phase symmetry indices, jitter, shimmer, noise-to-harmonic ratio, and maximum phonation time and qualitative parameters such as Voice Handicap Index, glottal gap, amplitude, and phase difference were used to evaluate the pre- and post-treatment status. RESULTS: In cases of vocal cord paralysis, vibratory changes of the vocal folds before and after intracordal injection could be identified immediately using 2D DKG. In overcorrection cases, all of the measured parameters were poor except for improvement of the glottal gap. In addition, 2D DKG showed appropriately the changes in vocal cord vibration before and after voice therapy in patients with presbyphonia. CONCLUSION: Two-dimensional DKG may be a useful diagnostic tool in evaluation of the vibratory characteristics of entire vocal cords. In addition, it may also play a role in providing a decision for treatment modalities.

Vibratory Characteristics of Diplophonia Studied by High Speed Video and Vibrogram Analysis.

Diplophonia can occur in patients with polyps, atrophy, paralysis, or scars. Its vibratory patterns have not been well characterized. High-speed video (HSV) analysis can contribute to their understanding. Twenty subjects with a diplophonic voice quality were studied by HSV. Diplophonia was due to medical causes including vocal fold paresis (n = 7), vocal atrophy (n = 5), polyps (n = 5), and scars/sulci (n = 3). The HSV was analyzed using a multislice digital videokymography (DKG). The DKG tracing was analyzed qualitatively and then transformed into a vibrogram waveform signal for frequency analysis. RESULTS: Vibratory abnormalities seen on HSVs explained the diplophonia. Subharmonics to the fundamental frequency can be visualized by DKG. None could be resolved by stroboscopy. One can stratify diplophonia as symmetric or asymmetric based on the involvement of one or both vocal folds. Scars and atrophy showed symmetric subharmonic production with ectopic beats every 4-10 beats. Some subjects showed anterior and posterior independent vocal fold oscillators. Asymmetric causes of diplophonia are common in patients with paralysis. Two different oscillation frequencies of each vocal fold generate in and then out of phase interaction between the two sides. Vibrogram analysis documents the frequent presence of interharmonic energy peaks above the dominant fundamental frequency. Eighteen of the 20 subjects have obvious subharmonic peaks. CONCLUSION: Patients with diplophonia have vibratory abnormalities arising from the vocal folds. HSV and vibrogram analysis followed by frequency analysis of the vibrogram can resolve vibratory abnormality into symmetric versus asymmetric causes and can document the type of vibratory abnormality.

Evaluation of clinical value of videokymography for diagnosis and treatment of voice disorders.

This study aimed at determining the clinical value of videokymography (VKG) as an additional tool for the assessment of voice disorders. 105 subjects with voice disorders were examined by an experienced laryngologist. A questionnaire was used to specify diagnosis, diagnostic confidence, and treatment recommendations before and after VKG. The first part of questionnaire was filled by the laryngologist for each patient after routine ear-nose-throat evaluation, including stroboscopy, the second part after the subsequent VKG examination. In 31% of subjects VKG confirmed the stroboscopic diagnosis, in 44% it made the diagnosis more accurate, in 20% there was adjustment of the treatment, and in 5% it was not found diagnostically useful. After VKG the diagnostic confidence increased in 68% of the subjects. VKG may help clinicians to take some important treatment decisions and may be recommended to be performed in patients, where clinicians are uncertain about diagnosis and treatment.

Comparing Chalk With Cheese-The EGG Contact Quotient Is Only a Limited Surrogate of the Closed Quotient.

The electroglottographic (EGG) contact quotient (CQegg), an estimate of the relative duration of vocal fold contact per vibratory cycle, is the most commonly used quantitative analysis parameter in EGG. The purpose of this study is to quantify the CQegg's relation to the closed quotient, a measure more directly related to glottal width changes during vocal fold vibration and the respective sound generation events. Thirteen singers (six females) phonated in four extreme phonation types while independently varying the degree of breathiness and vocal register. EGG recordings were complemented by simultaneous videokymographic (VKG) endoscopy, which allows for calculation of the VKG closed quotient (CQvkg). The CQegg was computed with five different algorithms, all used in previous research. All CQegg algorithms produced CQegg values that clearly differed from the respective CQvkg, with standard deviations around 20% of cycle duration. The difference between CQvkg and CQegg was generally greater for phonations with lower CQvkg. The largest differences were found for low-quality EGG signals with a signal-to-noise ratio below 10 dB, typically stemming from phonations with incomplete glottal closure. Disregarding those low-quality signals, we found the best match between CQegg and CQvkg for a CQegg algorithm operating on the first derivative of the EGG signal. These results show that the terms "closed quotient" and "contact quotient" should not be used interchangeably. They relate to different physiological phenomena. Phonations with incomplete glottal closure having an EGG signal-to-noise ratio below 10 dB are not suited for CQegg analysis.

Real-time Simultaneous DKG and 2D DKG Using High-speed Digital Camera.

INTRODUCTION: For the evaluation of voice disorders, direct observation of vocal cord vibration is important. Among the various methods, laryngeal videostroboscopy (LVS) is widely used, but it was not a true image because it collects images from different cycles. In contrast, high-speed videoendoscopy and videokymography have much higher frame rates and can assess functional and mobility disorders. OBJECTIVE: The purpose of the study is to describe real-time, simultaneous digital kymography (DKG), two-dimensional scanning (2D) DKG, and multi-frame (MF) LVS system using a high-speed digital camera, and identify the efficacy of this system in evaluating vibratory patterns of pathologic voice. METHODS: The pattern of vocal fold vibration was evaluated in a vocally healthy subject and in subjects with vocal polyp, vocal nodules, vocal cord scar, and vocal cord paralysis. We used both quantitative (left-right phase symmetry, amplitude symmetry index) and qualitative (anterior-posterior phase symmetry) parameters for assessment of vocal fold vibration. RESULTS: Our system could record videos within seconds and required relatively little memory. The speed of replay in the DKG, 2D DKG, MF LVS, and high-speed videoendoscopy was controllable. The number of frame per cycle with MF LVS was almost the same as the fundamental frequency. CONCLUSION: Our system can provide images of various modalities simultaneously in real time and analyze morphological and functional vibratory patterns. It can be possible to provide a greater level of information for the diagnosis and treatment of vibratory disorders.

Automated Multi-Peak Tracking Kymography (AMTraK): A Tool to Quantify Sub-Cellular Dynamics with Sub-Pixel Accuracy.

Kymographs or space-time plots are widely used in cell biology to reduce the dimensions of a time-series in microscopy for both qualitative and quantitative insight into spatio-temporal dynamics. While multiple tools for image kymography have been described before, quantification remains largely manual. Here, we describe a novel software tool for automated multi-peak tracking kymography (AMTraK), which uses peak information and distance minimization to track and automatically quantify kymographs, integrated in a GUI. The program takes fluorescence time-series data as an input and tracks contours in the kymographs based on intensity and gradient peaks. By integrating a branch-point detection method, it can be used to identify merging and splitting events of tracks, important in separation and coalescence events. In tests with synthetic images, we demonstrate sub-pixel positional accuracy of the program. We test the program by quantifying sub-cellular dynamics in rod-shaped bacteria, microtubule (MT) transport and vesicle dynamics. A time-series of E. coli cell division with labeled nucleoid DNA is used to identify the time-point and rate at which the nucleoid segregates. The mean velocity of microtubule (MT) gliding motility due to a recombinant kinesin motor is estimated as 0.5 µm/s, in agreement with published values, and comparable to estimates using software for nanometer precision filament-tracking. We proceed to employ AMTraK to analyze previously published time-series microscopy data where kymographs had been manually quantified: clathrin polymerization kinetics during vesicle formation and anterograde and retrograde transport in axons. AMTraK analysis not only reproduces the reported parameters, it also provides an objective and automated method for reproducible analysis of kymographs from in vitro and in vivo fluorescence microscopy time-series of sub-cellular dynamics.

Kymographic Analysis of Transport in an Individual Neuronal Sensory Cilium in Caenorhabditis elegans.

Intraflagellar Transport (IFT) is driven by molecular motors that travel upon microtubule-based ciliary axonemes. In the single-celled alga Chlamydomonas reinhardtii, movement of a single anterograde IFT motor, heterotrimeric kinesin-II, is required to generate two identical motile flagella. The function of this canonical anterograde IFT motor is conserved among all eukaryotes, yet multicellular organisms can generate cilia of diverse structures and functions, ranging from simple threadlike non-motile primary cilia to the elaborate cilia that make up rod and cone photoreceptors in the retina. An emerging theme is that additional molecular motors modulate the canonical IFT machinery to give rise to differing ciliary morphologies. Therefore, a complete understanding of the trafficking of ciliary receptors, as well as the biogenesis, maintenance, specialization, and function of cilia, requires the characterization of motor molecules.Here, we describe in detail our method for measuring the motility of proteins in cilia or dendrites of C. elegans male-specific CEM ciliated sensory neurons using time-lapse microscopy and kymography of green fluorescent protein (GFP)-tagged motors, receptors, and cargos. We describe, as a specific example, OSM-3::GFP puncta moving in cilia, but also include (Fig. 1) with settings that have worked well for us measuring movement of heterotrimeric kinesin-II, IFT particles, and the polycystin TRP channel PKD-2.

Live-cell imaging of neurofilament transport in cultured neurons.

Neurofilaments, which are the intermediate filaments of nerve cells, are space-filling cytoskeletal polymers that contribute to the growth of axonal caliber. In addition to their structural role, neurofilaments are cargos of axonal transport that move along microtubule tracks in a rapid, intermittent, and bidirectional manner. Though they measure just 10nm in diameter, which is well below the diffraction limit of optical microscopes, these polymers can reach 100 µm or more in length and are often packed densely, just tens of nanometers apart. These properties of neurofilaments present unique challenges for studies on their movement. In this article, we describe several live-cell fluorescence imaging strategies that we have developed to image neurofilament transport in axons of cultured neurons on short and long timescales. Together, these methods form a powerful set of complementary tools with which to study the axonal transport of these unique intracellular cargos.

Axonal actin in action: Imaging actin dynamics in neurons.

Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

Live cell imaging of cytoplasmic dynein movement in transfected embryonic rat neurons.

Live cell imaging of the movement of various membrane-bounded organelle cargos has enhanced our understanding of their function. Eukaryotic cells utilize microtubules and two classes of microtubule-based motor proteins, cytoplasmic dynein and members of the kinesin family, to deliver a variety of membrane-bounded organelles and other cargos to their appropriate locations. In order to better understand the functions and regulation of cytoplasmic dynein, we developed a method to study its location and motility in living cells. The technique takes advantage of the long thin axons of cultured hippocampal neurons. We use calcium phosphate to transfect fluorescent-tagged dynein intermediate chain (IC) subunits (DYNC1I) into cultured neurons. When the ICs are expressed at low levels, they are effective probes for the location of the cytoplasmic dynein complex in axons when living cells are imaged with fluorescence microscopy. The fluorescent subunit probes can be used to identify specific cargos of dynein complexes with different IC isoforms as well as the kinetic properties of cytoplasmic dynein.

Live-cell imaging of retrograde transport initiation in primary neurons.

Axonal transport is an essential function in neurons, as mutations in either motor proteins or their adaptors cause neurodegeneration. While some mutations cause a complete block in axonal transport, other mutations affect transport more subtly. This is especially true of mutations identified in human patients, many of which impair but do not block motor function in the cell. Dissecting the pathogenic mechanisms of these more subtle mutations requires assays that can tease apart the distinct phases of axonal transport, including transport initiation, sustained/regulated motility, and cargo-specific sorting or delivery. Here, we describe a live-cell photobleaching assay to assess retrograde flux from the distal axon tip, a measure for distal transport initiation. We have previously used this method to show that the CAP-Gly domain of DCTN1 is required for efficient retrograde transport initiation in the distal axon, but it is not required to maintain retrograde flux along the mid-axon (Moughamian & Holzbaur, 2012). This approach has allowed us to examine the effects of disease-causing mutations in the axonal transport machinery, and in combination with other assays, will be useful in determining the mechanisms and regulation of axonal transport in normal and diseased conditions.