Online analysis for calcium imaging with fluorescence adaptive optics scanning laser ophthalmoscopes
The analysis software anticipates your experiment directory follows the lab's conventions. Your experiment folder should be arranged as follows:
\Analysis- temporary files containing the transformations and file conversions will be saved here. After the experiment and online analysis is over, you are free to keep them for later use or discard them.\Ref- reflectance PMT channel, contains .txt files with stimulus information.\Vis- visible PMT channel, anticipated folder containing newly acquired and registered videos to analyzeTargetImage.tif- an image from a previous session for registration with your new data. Use one of the images output from Qiang's software if possible as the properties will most resemble the registered images produced during the experiment.TargetRois.txt- the ROIs from the target image in a label matrix.
Before running the first time, you need to let MATLAB know where FIJI is located on your computer
addpref('ao_tools', 'fiji_path', '...\Fiji.app\scripts')Replace '...\Fiji.app\scripts' with the full file path to the scripts folder within the FIJI.app folder for your ImageJ/FIJI installation.This is needed to establish the connection between MATLAB and ImageJ.
Open MATLAB and run the following line from the command line:
app = OnlineAnalysisApp();You will be prompted to select your experiment file, as described above.
Before importing data, go to the "Prefs" tab and ensure the settings under the header "Set before importing new data" are accurate. You only need the signal window if you intend to look at the frequency response. The background window dictates the F - F0 calculation.
Click Load New Video to load, register and analyze a new video. The registration quality is reported using the built-in ssim function. Once you have multiple epochs loaded, you can choose which epoch to view on the right list box. If there are stimuli associated with the videos, you can choose them to see an average of all epochs using that stimulus. Use the arrow keys to navigate through the ROIs and s to smooth or unsmooth the data. Press f to switch to frequency domain. If the keys don't work, click somewhere on the plot side of the window, then try again.
When you're done and want to disconnect from ImageJ, run from the command line:
ij.IJ.run("Quit", "");Important: A quirk of the software that communicates with MATLAB-ImageJ plugin is that it cannot be easily used from inside a function, only scripts or the command line. For this reason, it was necessary to occasionally use the base workspace. As long as you have the OnlineAnalysisApp open, don't delete the app variable you created when instantiating it.
- MATLAB 2015a or higher with the following toolboxes: Image Processing, Curve Fitting
- FIJI with the MATLAB-ImageJ plugin installed
Other than the requirements above, the faoslo-online package stands alone. The following open source toolboxes are included in the \lib folder: