Combinations of Patch-Clamp and Confocal Calcium Imaging in A calcium indicator is a fluorescent molecule that binds to Ca2+ ions patch-clamp, confocal imaging, and image processing and analysis (Du et al., ). Simultaneous electrophysiological and fluorescent imaging . Perform whole- cell patch clamp and calcium imaging recordings in Background subtraction and other calculations are performed using data analysis software. Calcium imaging, voltage imaging, calcium currents. Page 3. Abstract. The combination of fluorescence measurements of membrane potential and intracellular Ca. 2+ . approach of data analysis using the fit of the Ca. 2+ channels in voltage clamp experiments, the external solution also contained 1 µM.
and fluorescent Ca2+ imaging. analysis Patch-clamp
DIC is a powerful technique for studying live cells because it avoids the need for cell staining, preventing cytotoxic effects that may hinder natural cell functions. In the selection of an imaging device, the camera selected must be sensitive enough to detect the contrast differences created by using DIC. Additionally, the imaging sensor must have high responses across the visible wavelength range to detect the wide variety of voltage-sensitive and ion-sensitive dyes, which may be present in low concentrations.
Lastly, because having the ability to capture rapidly changing cellular activity is extremely important when publishing and presenting findings, the camera selected for the electrophysiology lab must be fast enough to capture the rapidly changing cellular activity.
Another contrast enhancement technique commonly used to image unstained samples in thick tissues is Dodt gradient contrast microscopy. This method uses a gradient of light to improve the details of a sample. Dodt gradient contrast microscopy uses a tube to segment off a section of incoming light and sends it through a screen diffuser.
The gradient light is collimated before interaction with the sample, which casts shadows of light on the edges of the sample. When several images are taken, shadows are cast along different areas of the field of view. The recombination of these images results in images with enhanced detail over any of the single images alone.
This technique is popular because it requires very little setup and can be used with many existing microscopes. The use of fluorescence microscopy is used extensively in many areas of electrophysiology. Fluorescent probes and transgenic dyes are often used to image and measure the influx of ions in the study of ion channels, as well as visualize specific areas of the cell Figure 6. Fluorescent labels can be used alone or with other dyes to display an image of two or more organelles overlaid over each other.
Two-photon fluorescence microscopy is commonly used with dyes with emission wavelengths in different regions of the visible spectrum. Valuable spatial information about the structure of labelled features can be collected with this method. Using two-photon microscopy techniques, exciting advancements have been reported in the literature, proving its usefulness in the study of neural networks and differentiation There are also several groups using novel materials to improve the ease of microscopy imaging, such as the introduction of fluorescent pipettes to improve accuracy of single-cell patching.
Studying how the electrical environment of the cell can affect the flow of ions and thus, the functionality of cell, is studied widely for its applicability to disease prevention and remediation. Knowledge of electrophysiological techniques as well as the common complications can lead to higher quality data generation.
Combining electrophysiology with other techniques such as optogenetics has contributed even further to the field of neuroscience and maintains the status of electrophysiology as a vital technique to our understanding of human biology. Log in to your account: Electrophysiology Shani Samuel, Field Applications Specialist, Photometrics Dan Croucher, Applications Team Manager, Photometrics Introduction Electrophysiology is a field of research that deals with the electrical properties of cells and biological tissues.
Electrophysiology Principle Researchers and clinicians use electrophysiology when studying the electrical properties of neural and muscle tissue. Ohm's law for potential difference calculation The membrane potential at equilibrium is also determined through experimentation.
The Nernst Equation for membrane voltage at equilibrium E is the potential Nernst potential for a given ion R is the universal gas constant and is equal to 8. Intracellular Electrophysiology Intracellular electrophysiology involves obtaining electrical conductivity information from across a single cell or junction. Extracellular Electrophysiology Some applications require obtaining conductivity and impulse information from a group of neighboring cells or tissues in a field known as Extracellular Electrophysiology.
SNr GABA neuron being patch clamped 6 One commonly used technique in electrophysiology studies involves the attachment of a micropipette to a cell s of interest Figure 1. Calcium Imaging Precise measurement and visualization of the action potential of a cell, tissue, or medium is an area of focus for many electrophysiologists.
Tissues were counterstained with the nuclear stain DAPI blue. Left Electrical stimulation results in excitation of all neurons in the local area. Middle Targeted optogenetic excitation of a single neuron. Right Targeted optogenetic inhibition of a single neuron 5 Optogenetics is a relatively new research technique that has gained a lot of popularity in electrophysiology communities due to its ability to target specific cells within a population. Concerns of the Electrophysiologist Series Resistance A tightly controlled electrophysiology laboratory monitors and aims to reduce resistance between the amplifier and the cell, tissue, or specimen used.
Noise Reduction Many disturbances in the measurement of the desired signal in an electrophysiology experiment can cause noise in the data. Electrophysiology noise sources and common remediation methods. Use signal filters Anti-vibration tables or micromanipulators Electrical isolation of charge carriers Dielectric noise Noise arising from loss of current through the capacitors of the system.
For solid dielectrics, use high-quality materials with a low dissipation factor Quartz, Sapphire, some ceramics. Careful selection of equipment, such as patch-clamp headstages. Remove high-loss dielectrics from the area. Shot noise Variations in the number of photons contacting the sensor as a function of the discrete nature of photons amplified at low photon counts.
Sensor cooling Pixel correction Averaging multiple images together Excess noise Electrode Noise — Noise associated with resistance in the electrode. Contributes to dielectric noise and thermal noise. Select low-noise patch-clamp pipette materials. Use only debris-free micropipettes and apply positive pressure before cell contact.
Vibrational Noise — Noise in the data collection due to small vibrations in the system or environment. Use an anti-vibration table. Seal Noise - Noise as a result of poor contact with cell membrane surface. Use pipette with appropriate aperture. If done by hand, improve laboratory techniques. Use robotic patch-clamping apparatus. See how this image was captured. Neuroscience and Calcium Imaging. Positioning the monopolar stimulating electrode. Furthermore, repositioning of the stimulating electrode may lead to movement of the slice and loss of recording.
The small release probability at the Schaffer collateral synapses may often compel one to record several times from the same set of spines in the hope of detecting EPSCaTs. This should be avoided as it increases the risk of photodamage and shortens the experiments. Care must be taken to limit the amount of excitation light and therefore scans on any individual spine. The quality of the slice and the whole-cell patch clamp recording. At Scientifica we take your privacy seriously and will only use your personal information to provide the products and services you have requested from us.
If you consent to us collecting and storing your data from this form please tick the checkbox below. The implementation of these techniques has been a powerful tool for our studies, and has allowed us to add to the vast amount of research surrounding neuronal calcium signaling.
Animal care and procedures met National Institutes of Health standards. Local animal care ethical standards must be adhered to. The authors declare that there are no conflicts of interest or competing interests. Your questions will be directed to the authors of the protocol.
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For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos. My bio page Reset the password. A high-quality database for basic life science protocols. Jianyang Du Erin Elizabeth Koffman. Original research article A brief version of this protocol appeared in: Mice are group housed before and during the experiment.
Isoflurane Henry Schein Animal Health, catalog number: C Glucose Fisher Scientific, catalog number: G Phosphocreatine Sigma-Aldrich, catalog number: Leica VT S Pipettes, single channel, 0. The mice are housed and bred in the animal facilities at the University of Toledo.
Application Note: Electrophysiology
Here we ask if gene analysis could be combined with electrophysiology to . calcium imaging, cells were injected with calcium-sensitive fluorescent dye Oregon. Fura-2 or Oregon Green BAPTA for in vivo calcium imaging? I'm trying to analyze the change of intracellular calcium level after Why is my fluorescence intensity excited at and following the same trend? Hi, I'm new to calcium imaging but have been doing electrophysiology (whole-cell patch clamping. Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor . used to perform patch-clamp analysis and intracellular calcium level to intracellular calcium changes and generates high resolution images for.