A Zeiss LSM 880 in the Fralin Imaging Center was used to collect the calcium imaging data. We acknowledge Dr. Michelle L Olsen and Yuhang Pan for their assistance with the IMARIS software. We acknowledge Dr. Lenwood S. Heath for constructive comments on the manuscript and Steven Giavasis for comments on the GitHub README file. This work was supported by NIH R21MH122987 (https://www.nimh.nih.gov/index.shtml) and NIH R01GM140130 (https://www.nigms.nih.gov/) to L.N. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Calcium imaging
<ol>
	<li>Fly larvae preparation 
	NOTE: Flies and larvae are maintained at 25 &#176;C under a 12 h:12 h light:dark cycle.
	<ol>
		<li>Anesthetize the flies with CO2. Sort 20-45 males and 20-45 females into each fly vial, and give them at least 24 h to 48 h to recover from the CO2 exposure. 
		NOTE: Fly exposure to CO2 should last for the shortest amount of time possible.</li>
		<li>To synchronize the larvae age, tap over the flies into new ...

<ol>
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This study developed a new ImageJ plugin, TACI, and described a workflow analyzing 3D calcium imaging. Many currently available tools focus on correcting the x/y motion, although motion on the z-axis also needs to be explicitly diagnosed or corrected6. During image acquisition in a live organism, movement on the z-axis is unavoidable even when the organism is immobilized, and some stimuli, such as temperature change, often cause significant z-drift. Increasing the height of the z-stacks will allow...

The level of intracellular calcium is a precise marker of neuronal excitability. Calcium imaging measures the changes in intracellular calcium to understand neuronal activity1. Studies in neuroscience have increasingly used this method due to the development of techniques for measuring intracellular calcium concentration, including genetically encoded calcium indicators (GECIs), such as GCaMP2,3, which can be noninvasively expressed in specific sets of neurons through genetic approaches. The lower costs of lasers and microscope components have also increased the use of calcium imaging4. Importantly, calcium imaging allows for recording and studying single neurons as well as large neuron populations simultaneously in freely moving animals5.
Nevertheless, the analysis of calcium imaging data is challenging because (1) it involves tracking the changes in fluorescence of individual cells over time, (2) the fluorescence signal intermittently disappears or reappears with neuronal responses, and (3) the neurons may move in all directions, specifically in and out of a focal plane or appearing on multiple planes4,6. Manual analysis is time-consuming and becomes impractical as the length of recordings and the number of neurons increases. Various software programs have been developed to accelerate the process of analyzing calcium imaging. Previously, software was designed in a limited experimental context, making it difficult for other laboratories to adopt it. Recent efforts to meet modern standards for software sharing have led to the development of several tools that can consistently analyze calcium imaging data across different groups7,8,9,10,11,12,13,14,15,16,17,18,19. However, most of these tools require programming knowledge and/or depend on commercial software. A lack of programming knowledge and software paywalls deter researchers from adopting these methods. Moreover, many of these tools focus on correcting the x/y motion, although motion on the z-axis also needs to be explicitly diagnosed and corrected6. There is a need for a computational tool to analyze 3D calcium imaging that focuses on neurons exhibiting z-drift and appearing on multiple z-planes. Ideally, this tool should use open-source software and not require programming knowledge to allow other laboratories to readily adopt it.
Here, we developed a new ImageJ plugin, TACI, to analyze 3D calcium imaging data. First, the software renames, if needed, and organizes the 3D calcium imaging data by z-positions. The cells of interest are tracked in each z-position, and their fluorescence intensities are extracted by TrackMate or other computational tools. TACI is then applied to examine the motion on the z-axis. It identifies the maximum value of a z-stack and uses it to represent a cell&#39;s intensity at the corresponding time point. This workflow is suited to analyzing 3D calcium imaging with motion in all directions and/or with neurons overlapping in the lateral (x/y) direction but appearing in different z-positions. To validate this workflow, 3D calcium imaging datasets from fly larval thermosensitive neurons and mushroom neurons in the brain were used. Of note, TACI is an open-source ImageJ plugin and does not require any programming knowledge.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blunt Fill Needel</td>
			<td>BD</td>
			<td>303129</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fisher Scientific&#160;</td>
			<td>10035-04-8</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Carbon dioxide</td>
			<td>Airgas</td>
			<td>UN1013</td>
			<td>Size 200 High Pressure Steel Cylinder</td>
		</tr>
		<tr>
			<td>CO2 bubbler kit</td>
			<td>Genesee</td>
			<td>59-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope LSM880</td>
			<td>Zeiss</td>
			<td>4109002107876000</td>
			<td>An inverted Axio Observer Z1, equipped with 5 lasers, 2 standard PMT detectors, 32-channel GaAsP dectectors, an Airyscan detector, and Definite Focus.2.</td>
		</tr>
		<tr>
			<td>DAQami software</td>
			<td>Measurement Computing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Genesee</td>
			<td>62-113</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Drosophila Agar</td>
			<td>Genesee</td>
			<td>66-111</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs, Inc.</td>
			<td>64-17-5</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Fly line: Ir21a-Gal4</td>
			<td>Dr. Paul Garrity lab</td>
			<td></td>
			<td>A kind gift</td>
		</tr>
		<tr>
			<td>Fly line: Ir21a-Gal80</td>
			<td>Dr. Lina Ni lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly line: Ir68a-Gal4</td>
			<td>Dr. Aravinthan DT Samuel lab</td>
			<td></td>
			<td>A kind gift</td>
		</tr>
		<tr>
			<td>Fly line: Ir93a-Gal4</td>
			<td>Dr. Paul Garrity lab</td>
			<td></td>
			<td>A kind gift</td>
		</tr>
		<tr>
			<td>Fly line: UAS-GCaMP6</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>42750</td>
			<td></td>
		</tr>
		<tr>
			<td>Flypad</td>
			<td>Genesee</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>General purpose forged brass regulator</td>
			<td>Gentec</td>
			<td>G152</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco PBS pH 7.4 (1x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Green Drosophila tubing</td>
			<td>Genesee</td>
			<td>59-124</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat transfer compound</td>
			<td>MG Chemicals</td>
			<td>860-60G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heatsink</td>
			<td>Digi-Key Electronics</td>
			<td>ATS2193-ND</td>
			<td>Resize to 12.9 x 5.5 cm</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>AmScope</td>
			<td>LED-6W</td>
			<td></td>
		</tr>
		<tr>
			<td>Inactive Dry Yeast</td>
			<td>Genesee</td>
			<td>62-108</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Pervical</td>
			<td>DR-41VL</td>
			<td>Light: dark cycle: 12h:12h; temperature: 25 &#176;C; humidity: 40-50% RH.</td>
		</tr>
		<tr>
			<td>Methyl-4-hydroxybenzoate</td>
			<td>Thermo Scientific</td>
			<td>126965000</td>
			<td>Fly food ingrediete</td>
		</tr>
		<tr>
			<td>Micro cover glass</td>
			<td>VWR</td>
			<td>&#160;48382-126</td>
			<td>22 x 40 mm</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-544-2</td>
			<td>25 x 75 x 1.0 mm</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Kleancolor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Drosophila vials</td>
			<td>Genesee</td>
			<td>32-113RL</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective&#160;</td>
			<td>Zeiss</td>
			<td>420852-9871-000</td>
			<td>LD LCI Plan-Apochromat 25x/0.8 Imm Corr DIC M27</td>
		</tr>
		<tr>
			<td>Peltier cooling module</td>
			<td>TE Technology</td>
			<td>TE-127-1.0-0.8</td>
			<td>30 x 30 mm</td>
		</tr>
		<tr>
			<td>Plugs</td>
			<td>Genesee</td>
			<td>49-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>Circuit Specialists</td>
			<td>CSI1802X</td>
			<td>10 volt DC 2.0 amp linear bench power supply</td>
		</tr>
		<tr>
			<td>Princeton Artist Brush Nepture</td>
			<td>Princeton Artist Brush Co.</td>
			<td></td>
			<td>Series 4750, size 2</td>
		</tr>
		<tr>
			<td>Sodium potassium L-tartrate tetrahydrate</td>
			<td>Thermo Scientific</td>
			<td>033241-36</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Stage insert&#160;</td>
			<td>Wienecke and Sinske</td>
			<td>432339-9030-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td>Any stereo microscope works</td>
		</tr>
		<tr>
			<td>T-Fitting</td>
			<td>Genesee</td>
			<td>59-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouple data acquisition device</td>
			<td>Measurement Computing</td>
			<td>USB-2001-TC</td>
			<td>Single channel</td>
		</tr>
		<tr>
			<td>Thermocouple microprobe</td>
			<td>Physitemp</td>
			<td>IT-24P&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Yellow Cornmeal</td>
			<td>Genesee</td>
			<td>62-101</td>
			<td>Fly food ingredient</td>
		</tr>
		<tr>
			<td>Z-axis piezo stage</td>
			<td>Wienecke and Sinske</td>
			<td>432339-9000-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

taci: an imagej plugin for 3d calcium imaging analysis

Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging is a widely used technique that requires sophisticated software to obtain reliable results, but many laboratories struggle to adopt computational methods when updating protocols to meet modern standards. Difficulties arise due to a lack of programming knowledge and paywalls for software. In addition, cells of interest display movements in all directions during calcium imaging. Many approaches have been developed to correct the motion in the lateral (x/y) direction.
This paper describes a workflow using a new ImageJ plugin, TrackMate Analysis of Calcium Imaging (TACI), to examine motion on the z-axis in 3D calcium imaging. This software identifies the maximum fluorescence value from all the z-positions a neuron appears in and uses it to represent the neuron&#39;s intensity at the corresponding t-position. Therefore, this tool can separate neurons overlapping in the lateral (x/y) direction but appearing&#160;on distinct z-planes. As an ImageJ plugin, TACI is a user-friendly, open-source computational tool for 3D calcium imaging analysis. We validated this workflow using fly larval thermosensitive neurons that displayed movements in all directions during temperature fluctuation and a 3D calcium imaging dataset acquired from the fly brain.

Workflow of 3D calcium imaging analysis 
In this study, we developed a new ImageJ plugin, TACI, and described a workflow to track z-drift and analyze 3D calcium imaging that pinpoints the responses of individual cells appearing in multiple z-positions (Figure 1). This tool has four functions: RENAME, ORGANIZE, EXTRACT,&#160;and MERGE. First, if the image names are not compatible with the ORGANIZ...

Watch this Scientific Journal Video about TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis at JoVE.com

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes.

Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging ...

During calcium imaging recording, cells move in all directions. Many tools have been developed to correct the X and Y motion, but motion on the Z axis also needs to be explicitly diagnosed and corrected. TACI is a user-friendly open source image J plug-in.It checks Z drift and analyzes 3D calcium imaging with motion in all directions. After preparing the fly larvae, rinse them in PBS three times. Pipette 75 microliters of PBS onto the center of a glass slide.Put one or two larvae in PBS and place the thermocouple micrprobe near the larvae. Cover the larvae and thermocouple microprobe with a glass cover slip and seal it with nail polish. Place the slide on the microscope stage.Find the focus using a 25 times objective and place the thermo electric cooler on the slide. In confocal software, focus on the fluorescent cells of interest. Adjust the laser power to avoid oversaturation and set the first and last slice positions in the Z stack setting.Simultaneously, start the Z stack scanning and temperature recording. Control the power supply that powers the Peltier to change the Peltier's surface temperature. Afterwards, stop the Z stack scanning and temperature recording.After downloading the TACI calcium imaging dot jar install the plugin in Fiji by clicking on plugins in the menu bar, followed by install in the dropdown menu. Then restart Fiji and run the plugin by clicking on plugins and choosing TACI calcium imaging. Choose the rename function to convert the TIFF file names to the required structure.Choose the folder in which the TIFF images need renaming by clicking browse folders and wait for the file name to be filled automatically Using the folder name. check the max T position and max Z position. Ensure that the parameter values for max T position and max Z position include all digits.Enter the position of each parameter in order. If the TIFF file names do not include the phrase and channel leave the corresponding parameter value blank and choose NA in order. If there's any posttext, added to the posttext parameter.Click rename to create a folder with the same name, followed by underscore R.Observe that the TIFF file names have been restructured in the folder to be compatible with the organize function. Next, use the organize function to save the TIFF images in the same Z position in one folder. Choose the folder in which the T images need organizing by clicking browse folders.If the parameter CSV file exists, wait for the parameter values to be filled in automatically. If the parameter CSV file does not exist, manually fill in the parameter values. Ensure that the parameter values of phase and channel include letters if present, while the parameter values of the T position and Z position should be the largest numbers of the T and Z positions.If the image file names do not include the phase or channel, enter NA.Then create gray scale TIFF images when needed by making sure that the box R images gray is unchecked. Click organize to create a folder with the same name followed by underscore gray underscore stacks, and to generate folders with the same name followed by underscore and the Z position number in the folder. Then observe that the TIFF files are sorted into corresponding folders by Z positions, and that a file named param dot CSV is generated in which the parameters and their values can be found.Now use trackmate in Fiji to extract the fluorescence intensities of the cells of interest from each Z position. Open the TIFF images in a Z position folder by Fiji and run TRACKMATE by clicking on plugins and selecting tracking, followed by Trackmate. Adjust the parameters using DOG or LOG detectors.Change the blob diameter threshold and median filter. Set the filters to remove the irrelevant signals. Set the linking max distance, gap closing max distance, and gap closing max frame gap.Export the fluorescence intensities of the regions of interest to a CSV file. For each neuron, create a folder and name it using the neuron number. Starting from neuron zero.Save the CSV files containing the fluorescence information of the corresponding neuron in the folder. Name the CSV files as mean intensity Z position number dot CSV and include at least two columns, position T and mean intensity, or mean intensity channel one. Create the background underscore list dot CSV file that contains the information of all neurons in a specific format, starting from neuron zero.Fill in the neuron numbers such as neuron zero in row one. Then provide the background intensity for each Z position analyzed. If four Z positions are analyzed for neuron zero, fill in four background intensity values below neuron zero.Save the created background list dot CSV file and folders containing the CSV files of the fluorescence information in one folder. Open TACI and choose the extract function. Then choose the folder by clicking on browse files.The background file is automatically filled in. Fill in the largest number of T positions for the number of T positions. Click extract to create a results folder including each neuron's CSV files and plots.The CSV files include the maximum fluorescence intensity, and delta F over delta zero at each T position. The plots are line charts of delta F over F zero versus T positions. Using the merge function, average the delta F over F zero from all neurons.Calculate the SEM and plot the average delta F over F zero over T positions. Choose the results folder created by the extract function by clicking on browse files. Fill in the number of T positions with the largest number of T positions.Click merge to create a merged data folder, including a merged underscored data CSV file and an average underscore DF underscore F zero dot PNG plot. The CSV file includes the average and SEM delta F over F zero information at each T position. The plot is a line chart of the average delta F over F zero over T positions.Calcium imaging of fly larval cool cells expressing GCaMP six M barely shows any fluorescence in the inactive state at 27 degrees Celsius. However, the intracellular calcium levels rapidly increased at 10 degrees Celsius. The calcium levels rapidly dropped when the temperature was increased.Calcium imaging of fly brain neurons expressing GCaMP six F at time 0.1 showed fluorescence in four of the seven neurons, and the intensity of these neurons decreased over time. In the presence of octinal, multiple neurons brightened. The fluorescence of 10 neurons increased simultaneously at time point 92, suggesting that these mushroom neurons respond to octinal odor.TACI can separate overlapping cells. In this maximal projection image, two overlapping neurons were identified. These neurons got separated in the orthro view, indicating that these neurons appeared in different Z positions.The orange cell showed the strongest signal on Z seven while the blue cell had the strongest signal on Z 10. The change in fluorescence these two cells and revealed the delayed but strong activation of the orange cell. During extraction, the maximum fluorescence value from all the Z positions in which a neuron appears is used to represent the neuron's intensity at the corresponding deposition.If using TACI to analyze a large number of neurons, some registration across Z positions needs to be included in a new function to be developed organized with fluorescence information. TACI provides a user-friendly, open source computational approach for 3D capsule imaging analysis to check cell motion on the Z axis when individual cells appear in multiple Z positions.

taci-an-imagej-plugin-for-3d-calcium-imaging-analysis

Research

JoVE Journal

Neuroscience

3.4K ビュー.  Virginia Polytechnic Institute and State University. カルシウムイメージングの記録中、細胞はあらゆる方向に移動します。XとYの動きを補正するために多くのツールが開発されていますが、Z軸の動きも明示的に診断して修正する必要があります。TACIは、ユーザーフレンドリーなオープンソースの画像Jプラグインです。Zドリフトをチェックし、全方向の動きで3Dカルシウムイメージングを分析します。ハエの幼虫を?...

ビデオ: TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes.

leafj: an imagej plugin for semi-automated leaf shape measurement

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

Demonstration of key methods for high throughput leaf measurements.  These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf...

outer-boundary assisted segmentation and quantification of trabecular bones by an imagej plugin

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial...

clock scan protocol for image analysis: imagej plugins

Clock Scan Protocol for Image Analysis: ImageJ Plugins

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software...

automated quantification and analysis of cell counting procedures using imagej plugins

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter.  Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the...

calcium imaging of drosophila ex vivo brain for epileptiform analysis

Calcium Imaging of Drosophila Ex Vivo Brain for Epileptiform Analysis  

Calcium Imaging of Drosophila Ex Vivo Brain for Epileptiform Analysis

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse...

catheterization of an anesthetized sheep for 3d angiogram analysis

Catheterization of an Anesthetized Sheep for 3D Angiogram Analysis

single-cell calcium imaging for studying the activation of calcium ion channels

Author Spotlight: Advancing Calcium-Related Ion Channel Studies Employing Imaging Techniques

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research...

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

automated analysis of intracellular phenotypes of salmonella using imagej

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and...

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

calcium imaging in electrically stimulated flat-mounted retinas

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

https://cloudfront.jove.com/CDNSource/teasers/65705_b.jpg

calcium imaging in mouse superior colliculus

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent...

an imaging technique to study the calcium response in bacteria-infected cells

This video demonstrates a technique for detecting local and global calcium ion response upon Shigella infection of human epithelial cells. Shigella invades host cells and releases invasion effectors, impacting intracellular calcium levels. The amplitude and frequency of the local and global cytosolic calcium are studied using a fluorescent...

An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells

ex vivo calcium imaging for drosophila model of epilepsy

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular...

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

brainbeats as an open-source eeglab plugin to jointly analyze eeg and cardiovascular signals

Author Spotlight: Advancing the Study of Brain-Heart Interplay with a Comprehensive EEGLAB Plugin for Multimodal Signal Analysis

The BrainBeats toolbox is an open-source EEGLAB plugin designed to jointly analyze EEG and cardiovascular (ECG/PPG) signals. It includes heartbeat-evoked potentials (HEP) assessment, feature-based analysis, and heart artifact extraction from EEG signals. The protocol will aid in studying brain-heart interplay through two lenses (HEP and features), enhancing reproducibility and...

BrainBeats as an Open-Source EEGLAB Plugin to Jointly Analyze EEG and Cardiovascular Signals

single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

analysis of beta-cell function using single-cell resolution calcium imaging in zebrafish islets

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium...

preparation of acute subventricular zone slices for calcium imaging

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described.  The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye...

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

Monitoring Epileptiform Events in &lt;em&gt;Drosophila&lt;/em&gt; via Calcium Imaging

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...