Hair cell calcium imaging software

In vivo calcium imaging of lateralline hair cells in. Otoferlin depletion results in abnormal synaptic ribbons and. Calcium imaging is an extremely useful technique for investigating the variety of roles that calcium ions have in functioning neurons. We present a comprehensive computational workflow for the analysis of neuronal population calcium dynamics. There are many reasons that ratio acquisition is selected as an imaging method. Theyll be releasing all of the information needed for making these devices yourself, including data analysis.

I have tried to look in the internet but cant understand what this technique actually is. Hardwareindependent software deploymentsystem imaging save time and simplify the installation of operating systems across your network by easily provisioning your pc fleet with the kace sda. Nod factor elicits two separable calcium responses inmedicago. When presented with 1 nm nod factor, root hair cells exhibited. Sensory neurons in the vomeronasal organ vno are thought to mediate a specialized olfactory response. Most of the hair cells in the saccular epithelium in vitro were electrically tuned to stimulation at 2550 hz. In this example the imaging software is programmed to send a 5 v ttl output during the time series image acquisition. Meaneya,c, adepartment of bioengineering, university of pennsylvania, united states bdepartment of cell biology and neuroscience, rutgers university, united states cdepartment of neurosurgery. Ionoptix calcium imaging system school of medicine. These signals provide a realtime, quantifiable readout of both mechanosensation and presynapsedependent calcium activities within these hair cells. An integrated calcium imaging processing toolbox for the. Calcium imaging childrens hospital of philadelphia. The calcium imaging research support laboratory provides the resources and expertise necessary to carry out stateoftheart intracellular calcium measurements in cultured or freshly isolated single cells. From beginners to experts, the analysis software lets you quantify subtle physiological changes and even labelfree samples with various graph options.

Calcium imaging calcium imaging was carried out as described previously 25,28 at near body temperature 3436 c. We examined the mutants using calcium imaging of acousticvibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials microphonics. Analyzing ion concentrations by measuring fluorophore shifts. In vivo calcium imaging through microendoscopic lenses enables imaging of previously inaccessible neuronal populations deep within the brains of freely moving animals.

Calcium ions generate a multitude of intracellular signals that control key functions, such as neurotransmitter release from synaptic vesicles. Protocols for culturing, plating, and maintaining cells will vary between different cell models however, the steps below outline a general workflow for cell based imaging assays from upstream media prep through downstream experimental analysis using a highcontent imaging system and cellular analysis software. Imaging setup for calcium imaging and haircell stimulation. One study even showed that imaging hair cells loaded with fura2 am. Trichoscan making hair growth measurable trichoscan. Calcium imaging of inner ear hair cells within the cochlear. This research has provided descriptions of the mechanosensory transduction mechanism, the membrane channels involved in frequency tuning, and the regulatory roles of intracellular calcium. Automates calcium ca spark analysis in confocal linescan images. Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using twophoton microscopy tao yuan baylor college of medicine, the bobby r. While other cell imaging systems often require harsh lighting and toxic dyes, live cell imagers are not constrained to these limitations and labs that perform this technique take measures to ensure the cells are maintained in a healthy state even while on the.

Here we have demonstrated the benefits of a livecell imaging based approach to measuring calcium dependent fluo4 am fluorescence using the lionheart fx automated live cell imager and gen 5 microplate reader and imager software. Cell imaging, cellular imaging analysis, cardiomyocytes. Mar 29, 2009 the hair cell was represented by a twodimensional array of three 0. Clearance is a hallmark of hair cell death in the lateral line, and provides a binary means to define a hair cell as surviving or dying when used in conjunction with timelapse imaging. Automated quantification of neuronal networks and single cell calcium dynamics using calcium imaging tapan p. In response to a pressureinduced deflection of stereocilia at the apical end of the cell. A major advantage of optical imaging of calcium signals is high spatial resolution. The axial step was increased in order to reduce the light dose impinging on the sample, while still obtaining an acceptable axial resolution.

Cellular structure for haircell mechanotransduction. In the case of observing levels of calcium calibration, invitrogens product information sheet for their calcium. Install software remotely and maintain uptodate gold master images across diverse hardware platforms. Targetedesterase induced dye loading ted supports the analysis of intracellular calcium store dynamics by fluorescence imaging. Hair advantage is hair style imaging software version 2. Afferents were clearly distinguished from efferent endings by the relative lack of small vesicles, less dense cytoplasm, and association with presynaptic vesicles clustered at the hair cell membrane, with or without a visible ribbon. In vivo calcium imaging of lateralline hair cells in larval zebrafish. The system can yield 30 ratiometric calcium images per second.

The bottom panel outlines the signals used to coordinate imaging with haircell stimulation. The ucla miniscope project is an nih brain initiativefunded project to open source headmounted calcium imaging devices. The aim of this study was the validation of the trichoscan method by comparative assessment of hair growth paramenters using trichoscan software versus manual identification of hairs prior to the final assessment of hair parameters. Hair advantage software is a professional hair style and makeup imaging system for the professional stylist. This system enables quick and easy quantitative calcium imaging without the need for specialized equipment or techniques. The system uses an intensified ccd camera and chopper wheel to rapidly switch between 340 and 380 nm excitation. The first measurement of the fertilization calcium signal was an imaging experiment in large 1 mm diameter fish eggs. In short, in this validation study showed an excellent correlation of hair growth parameters analyzed using. Pages in category cell imaging the following 43 pages are in this category, out of 43 total. Here, we characterize early calcium responses of wildtype and mutant medicago truncatula plants to nodulation factors produced by the bacterial symbiont sinorhizobium meliloti using a dualdye ratiometric imaging technique.

Automated quantification of neuronal networks and single. Control of hearing sensitivity by tectorial membrane calcium pnas. I will be very happy for recommendations for a good internet source. Try on thousands of hairstyles, hair colors and makeup. Light sheet fluorescence microscopy quantifies calcium. Tao yuan, simon shang gao, peter saggau, and john s. Cell tracking software tools laser scanning microscopy image analysis cell motility is a critical part of many important biological processes.

Imaging calcium entry sites and ribbon structures in two presynaptic cells david zenisek,1 viviana davila,2 lei wan,2 and wolfhard almers2 1department of cellular and molecular physiology, yale university school of medicine, new haven, connecticut 06520, and 2vollum institute, portland. Intracellular calcium imaging of hek293 cells was performed using a 384well fluorescent imaging plate reader flipr, molecular devices essentially as described 49. Recording population dynamics with cellular resolution is a unique strength of calcium imaging. In doing so, neuroscientists can investigate how neural activity may be linked to aspects of behaviour and cognition, effectively linking geneticallyidentified cells with function. Using fluorescence imaging and fluorescence correlation.

Cells were categorized as living or dying based on their fragmentation and clearance from the neuromast following 60 minutes of aminoglycoside exposure. The fluo4 calcium imaging kit has been formulated, optimized, and contains all. Direct imaging of er calcium with targetedesterase induced. Coordinated calcium signalling in cochlear sensory and non. During a postdoctoral fellowship with teresa nicolson at the vollum institute, she used a combination of scanning electron microscopy and in vivo calcium imaging to investigate the role of the primary cilium in developing hair cells figure 1. By combining in vivo calcium imaging with confocal and superresolution structured illumination microscopy, we found that genetic disruption or acute block of ca v 1.

Imaging calcium entry sites and ribbon structures in two. Monitoring calcium flux is an important method for characterizing gpcr activation. Fluo4 calcium imaging kit thermo fisher scientific. First, the sample was visualized using transmitted light that originated from underneath the sample. Outer hair cells ohcs are highly specialized sensory cells. Calcium imaging can be used to investigate phenomena across multiple spatial scales, from subcellular components such as dendritic spines all the way to populations of thousands of neurons. Conventional calcium imaging uses a calcium indicator dye loaded into cells. The software allows for background correction and easy calibration so that numerical values as well as images of calcium concentration in time can be obtained. Molecular probes, eugene, or through the patch pipette during wholecell recording. Alford department of otolaryngology, head and neck surgery, one baylor plaza, na102, houston, texas 77030. Ricci1,2 1department of otolaryngology, stanford university school of medicine, stanford, california. Enlargement of ribbons in zebrafish hair cells increases calcium currents but disrupts afferent spontaneous activity and timing of stimulus onset.

Magnetic nanoparticles for ultrafast mechanical control of. The official journal of the society for neuroscience. Suppression of inflammation delays hair cell regeneration. My research focuses on the physiology of hair cells isolated from the inner ear and studied with patch clamp recording and optical imaging. Calcium imaging of inner ear hair cells within the. I have exported out the time series data of all the cells in the field of view i. The fluo4 calcium imaging kit has been designed for the specific detection of calcium flux by imaging applications. Pdf calcium imaging of inner ear hair cells within the. Amplitude and time course of stereociliary calcium signals in an outer hair cell. The development of new imaging and optogenetics techniques to study the dynamics of large neuronal circuits is generating datasets of unprecedented volume and complexity, demanding the development of appropriate analysis tools.

The attachment of the cistern to the hair cell plasma membrane ranged from 0. Calcium signaling with flipr calcium 6 and 6qf assay kits on the flexstation 3 reader provides largest signal window of comparable calcium kits and dyes enables low signal screens, including endogenous, primary or stem cell targets. Spend more time focused on discovery with easy touse tools and educational materials for cell imaging, labeling, and detection. It will correct for motion in the movie, segment to find cells, and analysis on each active cell.

Integration of the precision of localizationbased superresolution methods in plant cell imaging including palm and storm is expected to expand and help us uncover details of the molecular architecture and intermolecular associations within complex subcellular structures in the near future. Virtual hairstyles hair imaging app free makeover software. Software package for analyzing one and twophoton calcium imaging datasets. The underlying functions can also be used to create guiless, command lineready analysis pipelines. Fast imaging of root hair calcium oscillations was done by recording 270 time points every 3 s, acquiring 15 planes spaced 3. Imaging revealed distinct populations of hair cells whose resonant response varied with the frequency of transepithelial electrical stimulation. I recently performed calcium imaging on some cells. A similar inference has been made from confocal imaging of bundle calcium signals in guinea pig isolated outer hair cells, although no information was provided about the size of the transducer. Calcium imaging is based on change in fluorescence emission spectra e. Monitoring intracellular calcium ion dynamics in hair cell. Uncaging for calciums role in cell cycle progression andor. Tmhs is an integral component of the mechanotransduction. Calcium imaging of inner ear hair cells within the cochlear epithelium of.

Jci mitochondrial calcium uptake underlies ros generation. These mutants are defective in balance and swim in circles but have no obvious morphological defects. The calcium signal took the form of a wave propagating from the point of sperm entry. We introduce cubic magnetic nanoparticles as an effective tool for precise and ultrafast control of mechanosensitive cells. Characterizing calcium mobilization using kinetic live cell. Mechanotransduction channels in hair cells are gated by tip links, which connect the stereocilia of a hair cell in the direction of their mechanical sensitivity.

Autocorrelation functions were computed by the zen software zeiss and. Combine powerful invitrogen imaging and detection reagents with evos cell imaging systems, thermo scientific microplate readers, and cellinsight high content analysis platforms. In the present work, a similar calcium imaging approach was used but the spatial resolution was enhanced by using mammalian cochlear inner hair cells where the step in height between rows of largediameter stereocilia allowed individual stereocilia to be imaged at. The rate of calcium influx in ipscderived cardiomyocytes was quantitated in a live cell kinetic assay using fluo4 calcium indicator with the invitrogen evos fl auto 2 imaging system using invitrogen celleste image analysis software. This study may provide a new idea for how to promote hair cell regeneration and functional recovery in adult mammals. Upon binding of calcium, a large increase 100fold in fluorescence emission can be detected. However, only one cell is examined at a time, and surrounding hair cells are damaged to gain. Calciuminduced calcium release supports recruitment of synaptic vesicles in auditory hair cells x manuel castellanomunoz, 1michael e. Localization of inner hair cell mechanotransducer channels. As their name implies, these imagers allow for visualization of living cells in a more natural state via time lapse photography. We also outline equipment required for calcium imaging and compare different imaging systems. Functional calcium imaging in zebrafish lateralline hair.

Cell tracking software tools laser scanning microscopy. The chapter focuses on calcium imaging in embryos, which is used to identify. Hair cells serve as the receptor cell for both the auditory and vestibular system of vertebrates 1,2. To minimize time to experimental results, this kit contains all of the reagents necessa. Hair cells are mechanosensors for the perception of sound, acceleration, and fluid motion. Calciuminduced calcium release during action potential. Conversely, activating channels reduced both synapticribbon size and the number of intact synapses. In the present work, a similar calcium imaging approach was used but the spatial resolution was enhanced by using mammalian cochlear inner hair cells where the step in height between rows of largediameter stereocilia allowed individual stereocilia to be imaged at the apical portion of the bundle.

This fiji analysis is adapted from previously established. Cell voyager 7000s cv7000s is a high content screening system with our original confocal scanner unit, a live cell stage incubator and a buildin liquid handler which enable long term live cell imaging and rapid kinetic analysis. Imaging calcium entry sites and ribbon structures in two presynaptic cells david zenisek,1 viviana davila,2 lei wan,2 and wolfhard almers2 1department of cellular and molecular physiology, yale university school of medicine, new haven, connecticut 06520, and 2vollum institute, portland, oregon 97201 we investigated the location of calcium entry sites and. Automated and sensitive cell tracking is essential to cell motility studies where the tracking results can be used for diagnostic or curative decisions and where mathematical models can be developed to. Zinc activates damagesensing trpa1 ion channels nature. Cellpathfinder is designed for our hca systems, cq1 and the cellvoyager series. Schematic of the imaging system requirements outlined in section 2.

Many fundamental functions of a cell strongly depend on delicate, but nevertheless dynamic balances of ions e. Upon restoration of tectorial membrane calcium, sensory cell function returns. Calcium imaging of single stereocilia in hair cells. However, the unit of interest is a cell, not a pixel, and typical calcium imaging experiments capture several to several dozen cells in one field of view. Advances in imaging plant cell dynamics plant physiology.

Includes a gui to allow users to do largescale batch analysis, accessed via the repositorys calciumimaginganalysis class. For all hair cell imaging experiments, a single hair cell was analyzed for each tgmyo6b. I am stimulating my cells in culture using a variety of stimuli and checking the response as calcium intensity changes, using live cell calcium imaging. In this chapter, we describe methods to perform functional calcium imaging in zebrafish lateralline hair cells. Raw calcium imaging data is in the form of a time series of images where the intensity of each pixel is the fluorescence intensity at that location grienberger and konnerth, 2012.

Zstacks were processed with imaris 3d software, where individual 2 m xz. I have encountered the term two photon calcium imaging in a few papers. Functional calcium imaging in zebrafish lateralline hair cells. Together, the two adaptation processes provide the hair cell with a highpass filter and the. We compare genetically encoded calcium indicators that have been used previously to measure calcium in lateralline hair cells. Calcium signaling in the cochlea cell communication and. Live cell calcium imaging of dissociated vomeronasal neurons. When the preparation was ready for imaging, the recording chamber was transferred to the microscope and held fixed in place on a rigid column.

The calcium calibration will determine the level of free calcium in um at each pixel in the dataset. Calcium signaling with flipr calcium 6 and 6qf assay kits. Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using twophoton microscopy. Oghalai calcium imaging of inner ear hair cells within the cochlear epithelium of mice using twophoton microscopy, journal of biomedical optics 151, 016002 1 january 2010. Modulation of intracellular calcium levels plays a key role in the transduction of many biological signals. We intensively modified the software to work with the hardware we. Monitoring intracellular calcium ion dynamics in hair cell populations.

Calciumdependent luminescence from the jellyfish protein aequorin was measured using an image intensifier. This means that they emit photons with different energy, thus have different emission wavelength. Gcamp6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateralline hair cells. Calcium imaging provides neuroscientists with the means to study specific populations of cells within or across brain regions in freelybehaving animals. Calciuminduced calcium release supports recruitment of. In the case of genetically encoded indicators expressed in lateralline hair cells, it is possible to examine the responses of all hair cells within a neuromast at once. For ion imaging, several fluorescent indicators sensor, construct, tracer, etc are available that have a change in quantum yield upon ion binding. Suppression of inflammation delays hair cell regeneration and.

The molecular constituents of the mechanotransduction channels of hair cells are not known. Kace system imaging and deployment for windows and mac. Widefield calcium imaging with calcium indicator fura2. Lastly, a method to visualize spatial calcium signals in the fiji software is described. Auditory hair cells reside in the inner ear, where they are interlaced in a precise pattern with various supporting cells. Every oasis implant system comes with mightexs image acquisition and analysis software that allows users to collect large data sets of neural recordings from calcium imaging experiments and provides the key processing and analysis tools required to help users quickly convert these large data sets into results that can be clearly interpreted. Calcium imaging laboratory rosalind franklin university. In this chapter, we will introduce the cellular structure that is responsible for hair cell mechanotransduction met.