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...

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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.

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3.4K visualizações.  Virginia Polytechnic Institute and State University. Durante o registro de imagens de cálcio, as células se movem em todas as direções. Muitas ferramentas foram desenvolvidas para corrigir o movimento X e Y, mas o movimento no eixo Z também precisa ser explicitamente diagnosticado e corrigido. TACI é um plug-in J de imagem de código aberto fácil de usar.Ele verifica a deriva Z e analisa imagens de cálcio 3D com movimento em todas as direções. Depois de preparar as larvas da mosca, enxágue-as em PBS três vezes. Pipeta 75 micr...