Quantifying Protein Dynamics by Kymograph Analysis.

Time-lapse imaging of the subcellular localization and dynamic behavior of proteins is critical to understand their biological functions in cells. With the advent of various methodologies and computational tools, the precise tracking and quantification of protein spatiotemporal dynamics have become feasible. Kymograph analysis, in particular, has been extensively adopted for the quantitative assessment of proteins, vesicles, and organelle movements. However, conventional kymograph analysis, which is based on a single linear trajectory, may not comprehensively capture the complexity of proteins that alter their course during intracellular transport and activity. In this chapter, we introduced an advanced protocol for whole-cell kymograph analysis that allows for three-dimensional (3D) tracking of protein dynamics. This method was validated through the analysis of tip-focused endocytosis and exocytosis processes in growing tobacco pollen tubes by employing both the advanced whole-cell and classical kymograph methods. In addition, we enhanced this method by integrating pseudo-colored kymographs that enables the direct visualization of changes in protein fluorescence intensity with fluorescence recovery after photobleaching to advance our understanding of protein localization and dynamics. This comprehensive method offers a novel insight into the intricate dynamics of protein activity within the cellular context.

A Hands-on Guide to AmoePy - a Python-Based Software Package to Analyze Cell Migration Data.

Amoeboid cell motility is fundamental for a multitude of biological processes such as embryogenesis, immune responses, wound healing, and cancer metastasis. It is characterized by specific cell shape changes: the extension and retraction of membrane protrusions, known as pseudopodia. A common approach to investigate the mechanisms underlying this type of cell motility is to study phenotypic differences in the locomotion of mutant cell lines. To characterize such differences, methods are required to quantify the contour dynamics of migrating cells. AmoePy is a Python-based software package that provides tools for cell segmentation, contour detection as well as analyzing and simulating contour dynamics. First, a digital representation of the cell contour as a chain of nodes is extracted from each frame of a time-lapse microscopy recording of a moving cell. Then, the dynamics of these nodes-referred to as virtual markers-are tracked as the cell contour evolves over time. From these data, various quantities can be calculated that characterize the contour dynamics, such as the displacement of the virtual markers or the local stretching rate of the marker chain. Their dynamics is typically visualized in space-time plots, the so-called kymographs, where the temporal evolution is displayed for the different locations along the cell contour. Using AmoePy, you can straightforwardly create kymograph plots and videos from stacks of experimental bright-field or fluorescent images of motile cells. A hands-on guide on how to install and use AmoePy is provided in this chapter.

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.

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.

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.

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.

Reconstitution of microtubule-based motility using cell extracts.

Long-range intracellular transport of organelles driven by kinesin and dynein motor proteins depends on additional cellular factors including adaptors and scaffolding proteins. While single-molecule studies of the motility of purified motor proteins have been a powerful approach, these assays are not fully representative of the complex interactions that occur in a cellular environment. To gain insights into the functioning of adaptor proteins that work in concert with motors proteins, motility assays in cell extracts have been developed. These assays are an attractive means to begin to dissect the roles of additional factors in motor-driven transport. Further, this system can be easily manipulated to study this process in different physiological environments. Here we describe in vitro reconstitution of motor-driven motility along microtubules in cell extracts, followed by considerations for data analysis and how these assays can be powerful in informing our understanding of basic cellular processes.

Total internal reflection fluorescence microscopy of intraflagellar transport in Tetrahymena thermophila.

Live imaging has become a powerful tool in studies of ciliary proteins. Tetrahymena thermophila is an established ciliated model with well-developed genetic and biochemical approaches, but its large size, complex shape, and the large number of short and overlapping cilia, have made live imaging of ciliary proteins challenging. Here we describe a method that combines paralysis of cilia by nickel ions and total internal reflection microscopy for live imaging of fluorescent proteins inside cilia of Tetrahymena. Using this method, we quantitatively documented the intraflagellar transport in Tetrahymena.

Comparative analysis of vocal fold vibration using high-speed videoendoscopy and digital kymography.

OBJECTIVES: To compare high-speed videoendoscopy (HSV) and digital kymography (DKG) in the vocal fold vibration analysis of normophonic women with no vocal fold abnormalities. STUDY DESIGN: Prospective study comparing quantitative parameters extracted by HSV and DKG. METHODS: Eighteen normophonic women whose age ranged from 18 to 45 years participated in the study. The procedures comprised HSV and DKG of the medial line of the vocal folds. The parameters evaluated were fundamental frequency (F0), open quotient (OQ), and duration of vibration cycle phases (open phase, closed phase, opening phase, and closing phase). RESULTS: The F0 results of HSV and DKG were similar. However, significant differences were found in both duration of vibration cycle phases and OQ, indicating a longer open phase in the vocal fold vibration when this phase was measured by HSV. CONCLUSIONS: The results emphasize the need to set up different normative threshold values for both HSV and DKG.

Laryngoscopy, stroboscopy and other tools for the evaluation of voice disorders.

This article discusses and analyzes the diagnosis and management of voice disorders. Beginning with an insightful description of dysphonia as a sign and symptom rather than diagnosis, and an analysis of its unifying principles, the discussion continues with a review of evaluation, laryngoscopy, stroboscopy, and their respective advantages and disadvantages.

Proprioceptive coupling within motor neurons drives C. elegans forward locomotion.

Locomotion requires coordinated motor activity throughout an animal's body. In both vertebrates and invertebrates, chains of coupled central pattern generators (CPGs) are commonly evoked to explain local rhythmic behaviors. In C. elegans, we report that proprioception within the motor circuit is responsible for propagating and coordinating rhythmic undulatory waves from head to tail during forward movement. Proprioceptive coupling between adjacent body regions transduces rhythmic movement initiated near the head into bending waves driven along the body by a chain of reflexes. Using optogenetics and calcium imaging to manipulate and monitor motor circuit activity of moving C. elegans held in microfluidic devices, we found that the B-type cholinergic motor neurons transduce the proprioceptive signal. In C. elegans, a sensorimotor feedback loop operating within a specific type of motor neuron both drives and organizes body movement.

Kymographic imaging of laryngeal vibrations.

PURPOSE OF REVIEW: Kymographic imaging is a modern method for displaying and evaluating vibratory behaviour of the vocal folds which is crucial for voice production. This review summarizes the state of the art of this method, and focuses on the progress in this area within the last 5 years. RECENT FINDINGS: Videokymography, using a special videocamera, offers high-speed (video)kymographic images in real time, which is advantageous in daily clinical practice. Two other methods use software to create kymograms retrospectively: digital kymography processes high-speed videolaryngoscopic recordings and offers numerous research possibilities, whereas strobovideokymography processes videostroboscopic recordings, and its use is limited to regular vibration patterns. Current studies reveal that high-speed kymographic images allow more reliable visual evaluation of vibrations than by watching video recordings. Image analysis procedures have been advanced to quantify the vibration properties of the vocal folds. New information has been obtained on asymmetry, mucosal waves, irregularities, phonation onset, and nonlinear dynamic phenomena in voice disorders, as well as in singing. SUMMARY: High-speed kymography visualizes vibratory features which are not simply observable via traditional methods. It shows large potential in better understanding the functional origin of hoarseness and unsteady phonatory states. Further research in this area is envisioned.

Collagen injection for correcting vocal fold asymmetry: high-speed imaging.

OBJECTIVES: We hypothesized that high-speed digital imaging with videokymographic and laryngotopographic analysis would provide a quantitative method to evaluate the effect of collagen injection for the correction of asymmetric and irregular vocal fold vibration in unilateral vocal fold paralysis. METHODS: Videokymographic and laryngotopographic analysis was performed for high-speed digital recordings of vocal fold vibration for visualizing the glottal vibratory patterns, and for quantifying the frequency of vibration of each vocal fold, respectively, including comparisons between the paralyzed and normal vocal folds before and after surgery. This included prospective observations of 11 subjects with unilateral vocal fold paralysis (4 male, 7 female; mean +/- SD age, 67.1 +/- 12.0 years) using high-speed digital image analysis before and after collagen injection. RESULTS: Analysis of the laryngotopographs revealed 2 distinct frequencies of vibration for the paralyzed and contralateral vocal folds for 8 of the 11 subjects before surgery. After collagen injection, the vibration frequencies became identical, despite asymmetric vibration amplitudes. Asymmetric vibration amplitudes were also observed in the other 3 subjects before surgery, but the amplitudes became symmetric after collagen injection, despite a persistent phase shift. CONCLUSIONS: Asymmetric vibration in vocal fold paralysis was exemplified by differences in vibration frequency and amplitude between the vocal folds. The present study showed that after collagen injection, these aspects of vibratory patterns improved toward symmetry. This surgical procedure could improve the functional symmetry of the larynx for phonation.

High-speed digital imaging laryngoscopy of the neoglottis following supracricoid laryngectomy with cricohyoidoepiglottopexy.

OBJECTIVES: This study aimed to analyse vocal performance and to investigate the nature of the neoglottal sound source in patients who had undergone supracricoid laryngectomy with cricohyoidoepiglottopexy, using a high-speed digital imaging system. METHODS: High-speed digital imaging analysis of neoglottal kinetics was performed in two patients who had undergone supracricoid laryngectomy with cricohyoidoepiglottopexy; laryngotopography, inverse filtering analysis and multiline kymography were also undertaken. RESULTS: In case one, laryngotopography demonstrated two vibrating areas: one matched with the primary (i.e. fundamental) frequency (75 Hz) and the other with the secondary frequency (150 Hz) at the neoglottis. In case two, laryngotopography showed two vibrating areas matched with the fundamental frequency (172 Hz) at the neoglottis. The interaction between the two areas was considered to be the sound source in both patients. The waveform of the estimated volume flow at the neoglottis, obtained by inverse filtering analysis, corresponded well to the neoglottal vibration patterns derived by multiline kymography. These findings indicated that the specific sites identified at the neoglottis by the present method were likely to be the sound source in each patient. CONCLUSIONS: High-speed digital imaging analysis is effective in locating the sites responsible for voice production in patients who have undergone supracricoid laryngectomy with cricohyoidoepiglottopexy. This is the first study to clearly identify the neoglottal sound source in such patients, using a high-speed digital imaging system.

Depth-kymography of vocal fold vibrations: part II. Simulations and direct comparisons with 3D profile measurements.

We report novel direct quantitative comparisons between 3D profiling measurements and simulations of human vocal fold vibrations. Until now, in human vocal folds research, only imaging in a horizontal plane was possible. However, for the investigation of several diseases, depth information is needed, especially when the two folds act differently, e.g. in the case of tumour growth. Recently, with our novel depth-kymographic laryngoscope, we obtained calibrated data about the horizontal and vertical positions of the visible surface of the vibrating vocal folds. In order to find relations with physical parameters such as elasticity and damping constants, we numerically simulated the horizontal and vertical positions and movements of the human vocal folds while vibrating and investigated the effect of varying several parameters on the characteristics of the phonation: the masses and their dimensions, the respective forces and pressures, and the details of the vocal tract compartments. Direct one-to-one comparison with measured 3D positions presents-for the first time-a direct means of validation of these calculations. This may start a new field in vocal folds research.

Videokymography in voice disorders: what to look for?

OBJECTIVES: Kymographic imaging through videokymography has been recognized as a convenient, novel way to display laryngeal behavior, yet little systematic research has been done to map the relevant features displayed in such images. Here we have aimed at specification of these features to enable systematic visual characterization and categorization of vocal fold vibratory patterns in voice disorders. METHODS: A cross-sectional, descriptive design was used. We selected 45 subjects and extracted 100 videokymographic images from the archive of more than 7,000 videokymographic examinations of subjects with a wide range of voice disorders. The images showed a large variety of vocal fold vibratory behaviors during sustained phonations. We visually identified the prominent features that distinguished the vibration patterns across the images. RESULTS: We divided the findings into 10 feature categories. They included refined traditional features (eg, mucosal waves), as well as additional features that are obscured in strobolaryngoscopy (eg, different types of irregularities, left-right frequency differences, shapes of lateral and medial peaks, cycle aberrations). CONCLUSIONS: The variations in the identified features reveal different behavioral origins of voice disorders. The findings open new possibilities for objective documentation and for monitoring vocal fold behavior in clinical practice through kymographic imaging.

Strobokymographic and videostroboscopic analysis of vocal fold motion in unilateral superior laryngeal nerve paralysis.

The clinical diagnosis of superior laryngeal nerve paralysis (SLNp) is infrequently made, because of the heterogeneity of clinical presentations and laryngoscopic findings. Laryngeal electromyography (LEMG) can provide the definitive diagnosis of this abnormality. With increasing use of LEMG in clinical practice, this condition is now more frequently appreciated by otolaryngologists. A characteristic, but infrequently reported, videostroboscopic vocal fold motion termed Gegenschlagen ("dashing-against-each-other") has previously been described to occur in unilateral SLNp. We encountered such motion in a clinical case, which we subsequently verified as unilateral SLNp by means of LEMG. This characteristic glottic motion was then verified in an in vivo canine model of phonation after unilateral SLNp. Videostrobokymography was performed to generate kymograms that illustrated this vocal fold motion clearly. Kymograms of both human and canine subjects with SLNp demonstrated an undulating motion of the horizontally shifting glottic space as the medial edges of the vocal folds chased each other 90 degrees out of phase. As one vocal fold mucosal edge was opening, the other was closing, and this repeated motion appeared as vocal folds chasing or dashing against each other. Although not uniformly seen in all cases, this vocal fold motion appears to be unique to SLNp.

A new generation videokymography for routine clinical vocal fold examination.

OBJECTIVE: This study aims to introduce a new-generation videokymographic system, which provides simultaneous laryngoscopic and kymographic image, for routine clinical vocal fold examination. STUDY DESIGN: The authors explored a new imaging method for diagnosis and evaluation of voice disorders. METHODS: The new-generation videokymographic system includes two charge-coupled device image sensors, a color area image sensor, and a monochromic high-speed line-scan image sensor. The high-speed line-scan image sensor is used to capture the kymogram, and the color area image sensor is used to obtain the laryngoscopic image. The two images can be displayed simultaneously on a video monitor or stored in a standard video recorder. Three subjects with nonpathologic voice were investigated in detail with the new videokymographic system. RESULTS: The high-quality laryngoscopic image and kymogram can be used directly for clinical purposes with no further postprocessing. The scan position of the kymogram is always indicated in the laryngoscopic image, which provides feedback for the operator to easily locate the expected scanning position. All varieties of vocal fold vibration, including irregular vibrations, phonation onset and offset, can be observed with the presented method. The continuous kymogram of the vocal fold vibration can be retrieved from a kymographic image sequence for quantitative analysis. CONCLUSIONS: The new-generation videokymography provides a simple, quick means to investigate vocal fold vibration, especially for voice disorders. It can emerge as an important tool for routine clinical vocal fold examination.