Value of high-speed videoendoscopy as an auxiliary tool in differentiation of benign and malignant unilateral vocal lesions.

PURPOSE: The study aimed to assess the relevance of objective vibratory parameters derived from high-speed videolaryngoscopy (HSV) as a supporting tool, to assist clinicians in establishing the initial diagnosis of benign and malignant glottal organic lesions. METHODS: The HSV examinations were conducted in 175 subjects: 50 normophonic, 85 subjects with benign vocal fold lesions, and 40 with early glottic cancer; organic lesions were confirmed by histopathologic examination. The parameters, derived from HSV kymography: amplitude, symmetry, and glottal dynamic characteristics, were compared statistically between the groups with the following ROC analysis. RESULTS: Among 14 calculated parameters, 10 differed significantly between the groups. Four of them, the average resultant amplitude of the involved vocal fold (AmpInvolvedAvg), average amplitude asymmetry for the whole glottis and its middle third part (AmplAsymAvg; AmplAsymAvg_2/3), and absolute average phase difference (AbsPhaseDiffAvg), showed significant differences between benign and malignant lesions. Amplitude values were decreasing, while asymmetry and phase difference values were increasing with the risk of malignancy. In ROC analysis, the highest AUC was observed for AmpAsymAvg (0.719; p < 0.0001), and next in order was AmpInvolvedAvg (0.70; p = 0.0002). CONCLUSION: The golden standard in the diagnosis of organic lesions of glottis remains clinical examination with videolaryngoscopy, confirmed by histopathological examination. Our results showed that measurements of amplitude, asymmetry, and phase of vibrations in malignant vocal fold masses deteriorate significantly in comparison to benign vocal lesions. High-speed videolaryngoscopy could aid their preliminary differentiation noninvasively before histopathological examination; however, further research on larger groups is needed.

Atomic force microscope kymograph analysis: A case study of two membrane proteins.

Kymograph analysis is employed across the biological atomic force microscopy (AFM) community to boost temporal resolution. The method is well suited for revealing protein dynamics at the single molecule level in near-native conditions. Yet, kymograph analysis comes with limitations that depend on several factors including protein geometry and instrumental drift. This work focuses on conformational dynamics of difficult-to-study sparse distributions of membrane proteins. We compare and contrast AFM kymograph analysis for two proteins, one of which (SecDF) exhibits conformational dynamics primarily in the vertical direction (normal to the membrane surface) and the other (Pgp) exhibits a combination of lateral dynamics and vertical motion. Common experimental issues are analyzed including translational and rotational drift. Conformational transition detection is evaluated via kymograph simulations followed by state detection algorithms. We find that kymograph analysis is largely robust to lateral drift. Displacement of the AFM line scan trajectory away from the protein center of mass by a few nanometers, roughly half of the molecule diameter, does not significantly affect transition detection nor generate undue dwell time errors. On the other hand, for proteins like Pgp that exhibit significant azimuthal maximum height dependence, rotational drift can potentially produce artifactual transitions. Measuring the height of a membrane protein protrusion is generally superior to measurement of width, confirming intuition based on vertical resolution superiority. In low signal-to-noise scenarios, common state detection algorithms struggle with transition detection as opposed to infinite hidden Markov models. AFM kymography represents a valuable addition to the membrane biophysics toolkit; continued hardware and software improvements are poised to expand the method's impact in the field.

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.

Application of the HRES 5562 Camera Using the HSDI Technique in the Diagnosis of Glottal Insufficiencies in Teachers.

OBJECTIVE: Teachers are the largest group of professional voice users with predispositions to functional or organic changes in the larynx. The dysfunction of intrinsic muscles of the larynx frequently occurs which leads to changes in parameters of quality of voice (dysphonia). The aim of the study is the assessment of parameters of vocal folds vibrations, Mucosal Wave morphology, Glottal Closure Type and Open Quotient (OQ) using High Speed (HS) camera and High Speed Digital Imaging (HSDI) technique in teachers with disorders of voice quality classified in GRBAS scale in glottal insufficiencies. MATERIAL AND METHOD: The study included group of 50 teachers of both genders, working in primary and secondary education units for 15 years, without systemic diseases with disorders of quality of voice confirmed by the results in GRBAS scale assessment. Vocal folds vibrations were assessed with HS camera by R. Wolf and HSDI technique. Rigid endoscope with 90o optics by the same company was used in the study. Vocal folds vibrations were registered with the speed of 4000 frames/sec. Regularity, symmetry of vibrations, Mucosal Wave (MW) morphology as well as Glottal Closure Type were assessed. Numerical value of OQ was determined in anterior, middle and posterior segment of the glottis during phonation. RESULTS: Assessment of real vibrations of vocal folds revealed irregularity, mean asymmetry of vibrations, MW reduction as well as glottal insufficiency, the most frequently (96%), in the middle segment - type E according to European Laryngological Society, less frequently (4%) - type E+C. The highest value of OQ (average 0.98) was registered in the middle segment of the glottis. In GRBAS scale, significant dysphonia has been registered, classified as G3R3B3A3 without the features of voice strain - S0. CONCLUSION: In group of teachers with extensive professional experience, glottal insufficiency in the middle segment was registered the most frequently which was confirmed by high values of OQ in this region obtained with HSDI technique. What is more, irregularity, mean asymmetry and asynchrony of vibrations with MW reduction were registered which confirmed the existence of glottal insufficiency in this group. GRBAS scale was useful in preliminary evaluation of dysphonia and its severity, however, the diagnosis of its clinical form was possible only with visualization of the larynx and objective parameters obtained with HSDI.

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.

A Portable Smartphone-Based Laryngoscope System for High-Speed Vocal Cord Imaging of Patients With Throat Disorders: Instrument Validation Study.

BACKGROUND: Currently, high-speed digital imaging (HSDI), especially endoscopic HSDI, is routinely used for the diagnosis of vocal cord disorders. However, endoscopic HSDI devices are usually large and costly, which limits access to patients in underdeveloped countries and in regions with inadequate medical infrastructure. Modern smartphones have sufficient functionality to process the complex calculations that are required for processing high-resolution images and videos with a high frame rate. Recently, several attempts have been made to integrate medical endoscopes with smartphones to make them more accessible to people in underdeveloped countries. OBJECTIVE: This study aims to develop a smartphone adaptor for endoscopes, which enables smartphone-based vocal cord imaging, to demonstrate the feasibility of performing high-speed vocal cord imaging via the high-speed imaging functions of a high-performance smartphone camera, and to determine the acceptability of the smartphone-based high-speed vocal cord imaging system for clinical applications in developing countries. METHODS: A customized smartphone adaptor optical relay was designed for clinical endoscopy using selective laser melting-based 3D printing. A standard laryngoscope was attached to the smartphone adaptor to acquire high-speed vocal cord endoscopic images. Only existing basic functions of the smartphone camera were used for HSDI of the vocal cords. Extracted still frames were observed for qualitative glottal volume and shape. For image processing, segmented glottal and vocal cord areas were calculated from whole HSDI frames to characterize the amplitude of the vibrations on each side of the glottis, including the frequency, edge length, glottal areas, base cord, and lateral phase differences over the acquisition time. The device was incorporated into a preclinical videokymography diagnosis routine to compare functionality. RESULTS: Smartphone-based HSDI with the smartphone-endoscope adaptor could achieve 940 frames per second and a resolution of 1280 by 720 frames, which corresponds to the detection of 3 to 8 frames per vocal cycle at double the spatial resolution of existing devices. The device was used to image the vocal cords of 4 volunteers: 1 healthy individual and 3 patients with vocal cord paralysis, chronic laryngitis, or vocal cord polyps. The resultant image stacks were sufficient for most diagnostic purposes. The cost of the device including the smartphone was lower than that of existing HSDI devices. The image processing and analytics demonstrated the successful calculation of relevant diagnostic variables from the acquired images. Patients with vocal pathologies were easily differentiable in the quantitative data. CONCLUSIONS: A smartphone-based HSDI endoscope system can function as a point-of-care clinical diagnostic device. The resulting analysis is of higher quality than that accessible by videostroboscopy and promises comparable quality and greater accessibility than HSDI. In particular, this system is suitable for use as an accessible diagnostic tool in underdeveloped areas with inadequate medical service infrastructure.

Kymographic characteristics of voice in women with Polycystic ovary syndrome.

PURPOSE: Polycystic ovary syndrome (PCOS) is heterogenous condition with commonly associated symptoms include irregular menstrual cycle, hirsutism, baldness, adult acne, and weight gain There have been few attempts at profiling the voice characteristics of women with polycystic ovary syndrome. Videokymography enables to detect even subtle variationsin vocal fold vibrations. The aim of the present study was to study the videokymographic characteristics among women with PCOS. METHODS: A cross-sectional study was carried out among 50 women with and without PCOS diagnosed on ultrasonography. Videokymography was carried out and the characteristics were perceptually analyzed using a vocal fold kymographic rating scale. The analysis of the kymogram was done for the following characteristics; presence of vocal fold vibration,interference of surroundings, Cycle to cycle variability, left-right asymmetry,cycle aberrations and shape of lateral peaks. The kymographic images were obtained for all the participants of both the groups and a subjective consensus evaluation was done by two clinicians. The percentage of participants with the listed kymographic characteristics were tabulated. Chi Square test was also done to decide if there was a significant difference between the two groups for different kymographic features of vocal fold vibration. RESULTS: Six of the 25 women with PCOS were found to have abnormal kymographic features such as surrounding structural interference, presence of cycle to cycle variability and the shape of lateral peaks. CONCLUSION: Early detection of the vocal abnormalities in individuals with PCOS would help in the vocal rehabilitation especially for professional voice users.

High speed digital videolaringoscopy: evaluation of vocal nodules and cysts in women.

PURPOSE: To evaluate and compare the parameters of Digital kymography obtained through the High-speed Videolaryngoscopy of women without laryngeal disorders, of women with vocal fold nodules and of women with vocal cysts. METHODS: A cross-sectional observational study in which 60 women aged 18 years and 45 years were selected. Three study groups were formed: 20 women without laryngeal disorder forming the control group (Group 1), 20 women with diagnosis of vocal fold nodules forming Group 2 and 20 women with diagnosis of vocal cysts forming Group 3. Subsequently the participants were evaluated by High-speed Videolaryngoscopy for analysis and comparison of laryngeal images using Digital kymography. The laryngeal parameters processed by the program KIPS® were: minimum, maximum and mean opening; dominant amplitude of the left and right vocal folds; dominant frequency of the right and left vocal folds; and close. RESULTS: The analysis of Digital kymography suggests that the presence of the vocal fold nodules and the vocal cysts tend to restrict more to the maximum and minimum opening of the vocal fold and the dominant amplitude of the opening variation in the middle region of the glottis. CONCLUSION: Digital kymography parameters were similar in the presence of vocal fold nodules and vocal cysts lesions.

[Application of videostrobokymography in analyzing factors affecting voice after surgery].

Objective:To elucidate the value of videostrobokymography in the evaluation of the characteristics of vocal cord vibration after the surgery. Methods:To analyze the spectrum of the voice before and after the microsurgery of the vocal folds. Using the videostrobokymography to reveal the changes of vibration, especially in the cases with poor spectrum elevation but without obvious recurrence. Results:Two patients were with elevated vocal parameters; 2 patients were with partially elevated vocal parameters（one with Jitter decreased while others elevated; one with Shimmer decreased while others elevated）; the other 3 patients were with decreased vocal parameters. The videostrobokynmography showed the recurrence in 1 patient, but not in others. In 4 cases with elevated vocal parameters, the videostrobokymography showed cycle-to-cycle variability of the amplitude, absence of vibration of vocal fold, glottal axis shift and phase difference . Conclusion:There are certain value of videostrobokymography in analyzing the minor changes of characteristic of vibration of vocal fold.

High speed digital videolaringoscopy: evaluation of vocal nodules and cysts in women / Videolaringoscopia digital de alta velocidade: avaliação de nódulos e cistos de pregas vocais de mulheres

ABSTRACT Purpose To evaluate and compare the parameters of Digital kymography obtained through the High-speed Videolaryngoscopy of women without laryngeal disorders, of women with vocal fold nodules and of women with vocal cysts. Methods A cross-sectional observational study in which 60 women aged 18 years and 45 years were selected. Three study groups were formed: 20 women without laryngeal disorder forming the control group (Group 1), 20 women with diagnosis of vocal fold nodules forming Group 2 and 20 women with diagnosis of vocal cysts forming Group 3. Subsequently the participants were evaluated by High-speed Videolaryngoscopy for analysis and comparison of laryngeal images using Digital kymography. The laryngeal parameters processed by the program KIPS® were: minimum, maximum and mean opening; dominant amplitude of the left and right vocal folds; dominant frequency of the right and left vocal folds; and close. Results The analysis of Digital kymography suggests that the presence of the vocal fold nodules and the vocal cysts tend to restrict more to the maximum and minimum opening of the vocal fold and the dominant amplitude of the opening variation in the middle region of the glottis. Conclusion Digital kymography parameters were similar in the presence of vocal fold nodules and vocal cysts lesions.

RESUMO Objetivo Avaliar e comparar os parâmetros da videoquimografia digital obtidos pela videolaringoscopia de alta velocidade de mulheres sem alterações laríngeas, de mulheres com nódulos de prega vocal e de mulheres com cistos vocais. Método Estudo observacional transversal, no qual foram selecionadas 60 mulheres com idade entre 18 e 45 anos. Três grupos foram formados: 20 mulheres sem alterações laríngeas formando o grupo controle (Grupo 1), 20 mulheres com diagnóstico de nódulos nas pregas vocais formando o Grupo 2 e 20 mulheres com diagnóstico de cistos vocais formando o Grupo 3. Posteriormente, os participantes foram avaliados por Videolaringoscopia de alta velocidade para análise e comparação de imagens da laringe usando videoquimografia digital. Os parâmetros videoquimográficos avaliados pelo programa KIPS® foram: aberturas mínima, máxima e média; amplitudes da prega vocal direita e esquerda; frequências da abertura da prega vocal direita e esquerda; e fechamento. Resultados A análise da videoquimografia digital sugere que a presença dos nódulos e dos cistos de pregas vocais tendem a restringir a abertura máxima e média da prega vocal e a amplitude dominante da variação de abertura na região média da glote. Conclusão Os parâmetros da videoquimografia digital foram semelhantes na presença de nódulos nas pregas vocais e lesões de cistos vocais.

Kymographic characteristics of voice in women with Polycystic ovary syndrome

ABSTRACT Purpose Polycystic ovary syndrome (PCOS) is heterogenous condition with commonly associated symptoms include irregular menstrual cycle, hirsutism, baldness, adult acne, and weight gain There have been few attempts at profiling the voice characteristics of women with polycystic ovary syndrome. Videokymography enables to detect even subtle variations in vocal fold vibrations. The aim of the present study was to study the videokymographic characteristics among women with PCOS. Methods A cross-sectional study was carried out among 50 women with and without PCOS diagnosed on ultrasonography. Videokymography was carried out and the characteristics were perceptually analyzed using a vocal fold kymographic rating scale. The analysis of the kymogram was done for the following characteristics; presence of vocal fold vibration,interference of surroundings, Cycle to cycle variability, left-right asymmetry,cycle aberrations and shape of lateral peaks. The kymographic images were obtained for all the participants of both the groups and a subjective consensus evaluation was done by two clinicians. The percentage of participants with the listed kymographic characteristics were tabulated. Chi Square test was also done to decide if there was a significant difference between the two groups for different kymographic features of vocal fold vibration. Results Six of the 25 women with PCOS were found to have abnormal kymographic features such as surrounding structural interference, presence of cycle to cycle variability and the shape of lateral peaks. Conclusion Early detection of the vocal abnormalities in individuals with PCOS would help in the vocal rehabilitation especially for professional voice users.

Objective Measures of Stroboscopy and High-Speed Video.

Videostroboscopy and high-speed imaging is now an accepted way to evaluate laryngeal function in patients with voice disorders. In patients with neurolaryngological diseases such as tremor, laryngeal spasm, and paralysis, having an objective way to evaluate vocal function is desirable. Using digital imaging and analysis, both the videostroboscopy and the high-speed video can be analyzed to obtain relevant measures of vocal function. From the videostroboscopy, the montage of the glottal cycle derived from steady of vocal vibration can be analyzed by using edge tracking software to obtain the glottal area waveform. The waveform is an indication of the efficiency of the vocal folds in acting as an oscillator and gives direct information as to the open and closed phase, the symmetry of vocal fold vibration and the degree of amplitude contribution from each fold. High-speed video overcomes the deficiencies of stroboscopy by allowing for all voice gestures to be studies. Using digital kymography and analysis of the vibrogram, analysis of the onset of vocal fold oscillation, evaluation of diplophonia, tremor, and voice breaks becomes possible. Furthermore, analysis of the high-speed video kymograph tracing using signal analysis allow the investigator to evaluate the frequency and power relationships of vocal fold vibratory function in the normal and disordered state.

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.

In Vivo Visualization of Moving Synaptic Cargo Complexes within Drosophila Larval Segmental Axons.

Identifying moving synaptic vesicle complexes and isolating specific proteins present within such complexes in vivo is challenging. Here we detail a protocol that we have developed that is designed to simultaneously visualize the axonal transport of two fluorescently tagged synaptic vesicle proteins in living Drosophila larval segmental nerves in real time. Using a beam-splitter and split view software, larvae expressing GFP-tagged Synaptobrevin (Syb) and mRFP-tagged Rab4-GTPase or YFP-tagged Amyloid Precursor protein (APP) and mRFP-tagged Rab4-GTPase are imaged simultaneously using separate wavelengths. Merged kymographs from the two wavelengths are evaluated for colocalization analysis. Vesicle velocity analysis can also be done. Such analysis enables us to visualize the motility behaviors of two synaptic proteins present on a single vesicle complex and identify candidate proteins moving on synaptic vesicles in vivo, under physiological conditions.

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.

Visual and Automatic Evaluation of Vocal Fold Mucosal Waves Through Sharpness of Lateral Peaks in High-Speed Videokymographic Images.

INTRODUCTION: The sharpness of lateral peaks is a visually helpful clinical feature in high-speed videokymographic (VKG) images indicating vertical phase differences and mucosal waves on the vibrating vocal folds and giving insights into the health and pliability of vocal fold mucosa. This study aims at investigating parameters that can be helpful in objectively quantifying the lateral peak sharpness from the VKG images. METHOD: Forty-five clinical VKG images with different degrees of sharpness of lateral peaks were independently evaluated visually by three raters. The ratings were compared to parameters obtained by automatic image analysis of the vocal fold contours: Open Time Percentage Quotients (OTQ) and Plateau Quotients (PQ). The OTQ parameters were derived as fractions of the period during which the vocal fold displacement exceeds a predetermined percentage of the vibratory amplitude. The PQ parameters were derived similarly but as a fraction of the open phase instead of a period. RESULTS: The best correspondence between the visual ratings and the automatically derived quotients were found for the OTQ and PQ parameters derived at 95% and 80% of the amplitude, named OTQ95, PQ95, OTQ80 and PQ80. Their Spearman's rank correlation coefficients were in the range of 0.73 to 0.77 (P < 0.001) indicating strong relationships with the visual ratings. The strengths of these correlations were similar to those found from inter-rater comparisons of visual evaluations of peak sharpness. CONCLUSION: The Open time percentage and Plateau quotients at 95% and 80% of the amplitude stood out as the possible candidates for capturing the sharpness of the lateral peaks with their reliability comparable to that of visual ratings.