TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

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.