find out the issue is associated with matlab version.

when I tried to do a automated ROI detection, the program shows an error as follows:

"Unrecognized function or variable 'find_unsaturatedPixels'.

Error in preprocess_data (line 70)
P.pixels = find_unsaturatedPixels(Y); % pixels that do not exhibit saturation

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 365)
[P, Y] = preprocess_data(Y, p, options);

Error in matlab.apps.AppBase>@(source,event)executeCallback(appdesigner.internal.service.AppManagementService.instance(),app,callback,requiresEventData,event) (line 63)
newCallback = @(source, event)executeCallback(appdesigner.internal.service.AppManagementService.instance(), ...

Error while evaluating Button PrivateButtonPushedFcn."

Is this because my file has something that is not compatible with this analysis? What should I do to solve this?

I am getting the error in the screenshot when ever I run Automated ROI detection. If I add manual initiation refinement, the neuron is correctly found, but the following error comes up after. I am not sure why it is failing. I am using MATLAB 2022a, I also tried MATLAB 2021b. I tried Ezcalcium V 3.0.1, 3.0, and 2.1.3 and I tried both Windows and Mac. I am also using the file created by the motion correction function. That successfully works on this file. It is also 8-bit.

Thank you very much for this very intuitive software I love it!

I am wondering if there is a way to register my image using a red non functional channel acquired in parralel to the calcium Imaging (my cells express gcamp amd td-tomato). My calcium signal is really dim during baseline and I am not sure it can be used for registration.

In the output I would like to make sure which signal is actually the raw deltaF/F0. is it the F_raw?

Thank you

Hi,

I have encountered a problem while running roi refinement, can someone help me out with this issue?

Thanks!

Unrecognized field name "Z_mod".

Error in ez_roi_refine/view_roi (line 305)
plot(app.RawUIAxes, app.roidata.Z_mod(cur_roi, :))

Error in ez_roi_refine/ROIListBoxValueChanged (line 718)
view_roi(app)

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 470)
Error while evaluating ListBox PrivateValueChangedFcn.

Unrecognized function or variable 'frames'.

Error in ez_roi_refine/calc_roidata (line 238)
app.roiref.baseline_window = floor(frames * baseline_loc);

Error in ez_roi_refine/LoadDataButtonPushed (line 679)
calc_roidata(app)

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 427)
Error while evaluating Button PrivateButtonPushedFcn.

Hello,

I have a small problem with the ROI Detection. When I use the deconvolution method MCEM (CVX and SPGL1), I get the error message << Unrecognized function or variable 'P_merged'>>.

Furthemore, when the method MCMC is selected I end up with <<All of the input arguments must be of the same size and shape.
Previous inputs had size 1 in dimension 1. Input #3 has size 79>>

Cheers,

Antoine

At the momento of the analysis of a Calcium signaling expression video in the format .avi there is this error message:

Unable to perform assignment because the size of the left side is 512-by-512 and the size of
the right side is 512-by-512-by-3.

Error in read_file (line 63)
imData(:,:,i-sframe+1) = Y1;

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 337)
Y = read_file(filename);

Error in appdesigner.internal.service.AppManagementService/executeCallback (line 171)
callback(appOrUserComponent, event);

Error in matlab.apps.AppBase>@(source,event)executeCallback(ams,app,callback,requiresEventData,event) (line 62)
newCallback = @(source, event)executeCallback(ams, ...

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback
Error while evaluating Button PrivateButtonPushedFcn.

which doesn't allow me to perform the analysis. I tried everything, I tried to convert the video to TIFF format and to convert every image in the frame to index image format and nothing worked.

Is worth nothing that I'm currently working in Mac M1 and the version of Matlab is R2022b

Hello I am using Matlab 2020b - the ROI detection step appears to complete ok, but when I then open the newly generated file in the ROI refinement app, the ROIs shown in pink are the same that were identified in the ROI detection step, but the ROIs shown in black that I can step through in the ROI refinement app do not line up with these pink circles. It seems that the ROI center information is not correct saved in the file at the end of the ROI detection step is incorrect.

ROI detection final fig:

ROI refinement - black dot does not line up with a pink circle:

Hi, thanks for amazing and helpful app
I have frame problem in ROI refinement step.

Warning window says 'baseline window has too many frames to fit in baseline location. setting baseline window to maximum possible value as fallback'

how can i set baseline window value..?

Hello,

When I click Run ROI Detection I get this:

Undefined function 'strel' for input arguments of type 'char'.

Error in CNMFSetParms (line 301)
{strel('disk',1,0)}

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 344)
options = CNMFSetParms(... % Check CNMFSetParms.m for more details

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 335)
Error while evaluating Button PrivateButtonPushedFcn.

Then I tried to install the Image Processing Toolbox, as I have seen in other thread but a message pops up saying:

Error 339: Your MathWorks account login session has expired.

But I am actually logged in to the mathworks page.

Thanks,

Helena

Hi all,

Thanks for creating this useful toolbox. I have a 15 min two-photon recording and had no issues going through the motion correction, but did encounter an issue with the ROI detection. The motion corrected tiff file is 5.38 GB and contains 22,007 8-bit tiff images, but for some reason the code turns it into a 21.5 GB file when trying to run the ROI detection.

My RAM is 16 GB (at least 3x as large as the file I am working with) but of course if the file becomes 21 GB during the ROI detection processing step, my RAM memory is not big enough.

See below for the error message in Matlab:

ezcalcium
Starting ROI detection for C:\Users\Lilian Emming\Desktop\avi 4254\video_42554_mcor.tif
Requested 512x512x22007 (21.5GB) array exceeds maximum array size preference (15.8GB). This might cause MATLAB to become unresponsive.

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 338)
if ~isa(Y, 'single'); Y = single(Y); end % convert to single

Related documentation

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 410)
Error while evaluating Button PrivateButtonPushedFcn.

Any ideas why the file becomes 4x as large during this step?

Thanks!

Lilian

hi
I'm trying to run your code but I'm facing some issues...

1. Starting ROI detection for E:\My Drive\2P\Rachely\Data\allodynia\Burn_innocous\2021_08_19\Registered (Green)_Stack.tif
   Undefined function 'read_file' for input arguments of type 'char'.

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 337)
Y = read_file(filename);
there is no read_file function, so I had to change it to:
t = Tiff(filename,'r');
Y = read(t);

2. Error using CNMFSetParms (line 158)
   Ambiguous parameter name 'p' (pixels, plot_df, pause_time).

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 345)
options = CNMFSetParms(... % Check CNMFSetParms.m for more details

and I couldn't solve it.

thank you

Hi! When I was trying to load the .mat file in the ROI refinement module, here pops up the following error.
"Warning: Image is too big to fit on screen; displaying at 50%

In images.internal.initSize (line 71)
In truesize (line 48)
In ez_roi_refine>load_ROI_Callback (line 380)
In gui_mainfcn (line 95)
In ez_roi_refine (line 42)
In matlab.graphics.internal.figfile.FigFile/read>@(hObject,eventdata)ez_roi_refine('load_ROI_Callback',hObject,eventdata,guidata(hObject))
Error using reshape
To RESHAPE the number of elements must not change.

Error in ez_roi_refine>view_ROI_function (line 566)
single_ROI = full(reshape(handles.ROI.A_or(:,ROI_number),map_size(1),map_size(2)));

Error in ez_roi_refine>load_ROI_Callback (line 471)
view_ROI_function(hObject, eventdata, handles) %Automatically load first ROI

Error in gui_mainfcn (line 95)
feval(varargin{:});

Error in ez_roi_refine (line 42)
gui_mainfcn(gui_State, varargin{:});

Error in matlab.graphics.internal.figfile.FigFile/read>@(hObject,eventdata)ez_roi_refine('load_ROI_Callback',hObject,eventdata,guidata(hObject))

Error while evaluating UIControl Callback."

I can see the whole field of view and traces in the GUI, but the enlarged view of the current ROI is black and the currently selected ROI is not highlighted in black. I am wondering how shall I fix it?
Thanks for the help!

Hi

I got EZcalcium GUI but when I click the "motion correction" I got an error message as follow

Function 'getRunningApp' is not defined to 'ez_motion_correction' type ...

error: ez_motion_correction (line 666)
runningApp = getRunningApp(app);

error: ezcalcium/MotionCorrectionButtonPushed (line 23)
ez_motion_correction

error using:
matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 335)

Thanks and I will wait for your help

Hi I run into an issue when I import my file for the motion correction. It says

"Unable to perform assignment because the size of the left side is 480-by-720 and the
size of the right side is 480-by-720-by-3.

Error in read_file (line 63)
imData(:,:,i-sframe+1) = Y1;

Error in ez_motion_correction/RunMotionCorrectionButtonPushed (line 240)
FOV = size(read_file(filename, 1, 1));

Error in matlab.apps.AppBase>@(source,event)executeCallback(appdesigner.internal.service.AppManagementService.instance(),app,callback,requiresEventData,event) (line 63)
newCallback = @(source, event)executeCallback(appdesigner.internal.service.AppManagementService.instance(), ...

Error while evaluating Button PrivateButtonPushedFcn."

My original file is a MOV file with frame width of 720 and frame hight of 450. What should I do to make the file compatible with the program?

This seems like a great tool, especially for ROI detection. I've just begun trying to use it after installation of toolboxes and ran into an unexpected error message with the first AVI file I tried to load (see command window report below). The AVI input file is greyscale single channel, saved in Fiji from an image sequence of TIFF files.

Which component uses "find_unsaturatedPixels", and what might be going wrong?

_Starting ROI detection for C:\Users\bdoyle\OneDrive - Biogen\Desktop\2wk_Neuroburst_CoCulture_Well__D_007\New_Channel_0.avi
Unrecognized function or variable 'find_unsaturatedPixels'.

Error in preprocess_data (line 70)
P.pixels = find_unsaturatedPixels(Y); % pixels that do not exhibit saturation

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 365)
[P, Y] = preprocess_data(Y, p, options);

Error in matlab.apps.AppBase>@(source,event)executeCallback(appdesigner.internal.service.AppManagementService.instance(),app,callback,requiresEventData,event) (line 63)
newCallback = @(source, event)executeCallback(appdesigner.internal.service.AppManagementService.instance(), ...

Error while evaluating Button PrivateButtonPushedFcn._

Hello, when I try to load a .avi file I got this error:

Unable to perform assignment because the size of the left side is 256-by-256 and the size of the right side is
256-by-256-by-3.

Error in read_file (line 63)
imData(:,:,i-sframe+1) = Y1;

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 337)
Y = read_file(filename);

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 386)
Error while evaluating Button PrivateButtonPushedFcn.

I obtained the same error for both motion correction and ROI detection function.
Could you please help me with that ?

Thank you a lot in advance for your help.

Best.

hi ,is there anyone could explain why i get this message? thanks.

Analyzing and transferring files to the workers ...done.
Error using parallel.FevalFuture/fetchNext
The function evaluation completed with an error.

Error in normcorre_batch_even (line 258)
[idx, shifts_temp, Mf_temp] = fetchNext(future_results);

Error in ez_motion_correction/RunMotionCorrectionButtonPushed (line 275)
[~, ~, template, options_mc] = normcorre_batch_even(filename, options);

Caused by:
Error using register_frame (line 6)
Undefined function 'split_frame' for input arguments of type 'single'.

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 386)
Error while evaluating Button PrivateButtonPushedFcn.

Hi, could you explain more details about the output file of ROI detection? For example, what is F_raw? How does F_raw calculated.
And could you also output the average pixel value of each ROI? The real raw pixel value, not your processed F_raw? Thank you.

Hi,

I just dowloaded EZcalcium and I'm trying to see how it works. I am not a really Matlab person.
I'm using a 16-bit TIFF file previously converted from a .oir file. There is only 1 channel with 1500 frames.
The following errors appeared when applying the Motion Correction:

Error using parallel.FevalFuture/fetchNext
The function evaluation completed with an error.

Error in normcorre_batch_even (line 258)
[idx, shifts_temp, Mf_temp] = fetchNext(future_results);

Error in ez_motion_correction/RunMotionCorrectionButtonPushed (line 275)
[~, ~, template, options_mc] = normcorre_batch_even(filename, options);

Error in appdesigner.internal.service.AppManagementService/executeCallback (line 171)
callback(appOrUserComponent, event);

Error in matlab.apps.AppBase>@(source,event)executeCallback(ams,app,callback,requiresEventData,event) (line 62)
newCallback = @(source, event)executeCallback(ams, ...

Caused by:
Error using register_frame
Undefined function 'imwarp' for input arguments of type 'single'.

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback
Error while evaluating Button PrivateButtonPushedFcn.

Could you please tell me how to fix this errors?
Thank you in advance!

Elena

I'm encountering multiple errors (first two appear to be Out of Memory), and the third (Error using classify_comp_corr
Array construction from ByteBuffer Threw an exception).

The file I'm trying to analyze is is a 5,4GB tif with 22000 frames. I have a power desktop with 64GB RAM. Are these memory issues still something that should be expected?

Any advice on how to proceed with files like this? Should I divide the stack into multiple files and run ROI detection for those, and then use the ROI matching feature? It is important for me to keep track which neurons are active during various stages of the time-series.

----- ERROR 1

Starting ROI detection for D:\x\M5testavi_mcor.tif
Estimating the noise power for each pixel from a simple PSD estimate... done
No spatial downsampling is performed. Consider spatial downsampling if the field of view is very large.
starting resampling
Initializing components with greedy method
found 10 out of 200 neurons..
found 20 out of 200 neurons..
found 30 out of 200 neurons..
found 40 out of 200 neurons..
found 50 out of 200 neurons..
found 60 out of 200 neurons..
found 70 out of 200 neurons..
found 80 out of 200 neurons..
found 90 out of 200 neurons..
found 100 out of 200 neurons..
found 110 out of 200 neurons..
found 120 out of 200 neurons..
found 130 out of 200 neurons..
found 140 out of 200 neurons..
found 150 out of 200 neurons..
found 160 out of 200 neurons..
found 170 out of 200 neurons..
found 180 out of 200 neurons..
found 190 out of 200 neurons..
found 200 out of 200 neurons..
Refining initial estimates with HALS...Starting parallel pool (parpool) using the 'Processes' profile ...
Connected to the parallel pool (number of workers: 12).
done
Updated spatial components
146 out of 200 components updated
194 out of 200 components updated
199 out of 200 components updated
200 out of 200 components updated
146 out of 200 components updated
194 out of 200 components updated
199 out of 200 components updated
200 out of 200 components updated
Error using classify_comp_corr
Out of Memory during deserialization

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 391)
rval_space = classify_comp_corr(Y, A, C, b, f, options);

Error in appdesigner.internal.service.AppManagementService/executeCallback (line 171)
callback(appOrUserComponent, event);

Error in matlab.apps.AppBase>@(source,event)executeCallback(ams,app,callback,requiresEventData,event) (line 62)
newCallback = @(source, event)executeCallback(ams, ...

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback
Error while evaluating Button PrivateButtonPushedFcn.

----- ERROR 2

Warning: File: update_temporal_components.m Line: 234 Column: 40
The temporary variable 'model_ar' will be cleared at the beginning of each iteration of the parfor-loop. If 'model_ar' is used before it is set, a runtime error will occur. For more information, see Parallel for Loops in MATLAB,
"Uninitialized Temporaries".
147 out of 200 components updated
194 out of 200 components updated
200 out of 200 components updated
147 out of 200 components updated
194 out of 200 components updated
200 out of 200 components updated
Warning: A worker aborted during execution of the parfor loop. The parfor loop will now run again on the remaining workers.
Warning: A worker aborted during execution of the parfor loop. The parfor loop will now run again on the remaining workers.
Error using parallel.internal.pool.serialize
Out of Memory during serialization

Error in distcomp.remoteparfor/serialize (line 280)
data = parallel.internal.pool.serialize(...

Error in distcomp.remoteparfor/buildParforController (line 162)
serializedInitData = obj.serialize(obj.InitData, initDataBufSize);

Error in distcomp.remoteparfor/rebuildParforController (line 190)
obj.buildParforController();

Error in distcomp.remoteparfor/handleIntervalErrorResult (line 259)
obj.rebuildParforController();

Error in distcomp.remoteparfor/getCompleteIntervals (line 396)
[r, err] = obj.handleIntervalErrorResult(r);

Error in classify_comp_corr (line 116)
parfor i = 1:K_m

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 391)
rval_space = classify_comp_corr(Y, A, C, b, f, options);

Error in appdesigner.internal.service.AppManagementService/executeCallback (line 171)
callback(appOrUserComponent, event);

Error in matlab.apps.AppBase>@(source,event)executeCallback(ams,app,callback,requiresEventData,event) (line 62)
newCallback = @(source, event)executeCallback(ams, ...

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback
Error while evaluating Button PrivateButtonPushedFcn.

----- ERROR 3

ezcalcium
Starting ROI detection for D:\x\M5testavi_mcor.tif
Estimating the noise power for each pixel from a simple PSD estimate... done
No spatial downsampling is performed. Consider spatial downsampling if the field of view is very large.
starting resampling
Initializing components with greedy method
found 10 out of 200 neurons..
found 20 out of 200 neurons..
found 30 out of 200 neurons..
found 40 out of 200 neurons..
found 50 out of 200 neurons..
found 60 out of 200 neurons..
found 70 out of 200 neurons..
found 80 out of 200 neurons..
found 90 out of 200 neurons..
found 100 out of 200 neurons..
found 110 out of 200 neurons..
found 120 out of 200 neurons..
found 130 out of 200 neurons..
found 140 out of 200 neurons..
found 150 out of 200 neurons..
found 160 out of 200 neurons..
found 170 out of 200 neurons..
found 180 out of 200 neurons..
found 190 out of 200 neurons..
found 200 out of 200 neurons..
Refining initial estimates with HALS...Starting parallel pool (parpool) using the 'Processes' profile ...
Connected to the parallel pool (number of workers: 12).
done
Updated spatial components
Warning: File: update_temporal_components.m Line: 234 Column: 40
The temporary variable 'model_ar' will be cleared at the beginning of each iteration of the parfor-loop. If 'model_ar' is used before it is set, a runtime error will
occur. For more information, see Parallel for Loops in MATLAB, "Uninitialized Temporaries".
147 out of 200 components updated
194 out of 200 components updated
200 out of 200 components updated
147 out of 200 components updated
194 out of 200 components updated
200 out of 200 components updated
Error using classify_comp_corr
Array construction from ByteBuffer Threw an exception.

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 391)
rval_space = classify_comp_corr(Y, A, C, b, f, options);

Error in appdesigner.internal.service.AppManagementService/executeCallback (line 171)
callback(appOrUserComponent, event);

Error in matlab.apps.AppBase>@(source,event)executeCallback(ams,app,callback,requiresEventData,event) (line 62)
newCallback = @(source, event)executeCallback(ams, ...

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback
Error while evaluating Button PrivateButtonPushedFcn.

IdleTimeout has been reached.
Parallel pool using the 'Processes' profile is shutting down.

Hi, anytime I add .tif or .avi files to run ROI detection or any other function I receive the error Unable to perform assignment because the size of the left side is 1216-by-1920 and the size of the right side is 1216-by-1920-by-3. Could someone please help me fix this? Thanks

I work with iPSC neurons and I'm interested in ratio-ing active vs inactive neurons in a field of view. Ideally I would detect all neurons using EZcalciums tools, assess whether or not they spontaneously depolarized in the time of the recording, and then bin them accordingly (active vs inactive).

I had two questions:

1. After testing on a few AVIs and tiff stacks, I've found that EZcalcium tends to omit some of the non-depolarizing cells appearing in the field of view (cells that are stably fluorescent for our neuron specific calcium indicator, but no change in fluorescence). Is there anyway to overcome this through settings, or is depolarization and change in fluorescence over time a key component of the ROI selection algorithm? (is there any way to grab more inactive living neurons with automated ROI selection?)

2. In your experience, have you seen any significant differences between TIFF stack vs AVI input files? Does the loss of information from TIFF to AVI have an impact?

Thank you for your help,
Ben

Hi,

I am using your software to generate the calcium signal extraction. While doing this I receive the following signal:

As you can see here, the signal is negative in some sections. This doesn't make sense with the theory. Is there any technical reason behind this, or is it possible to fix the signal to have only positive values?

Best regards,

Hello just want to try you your program and so far have not been able to get to to run. I see the really nice GUI and am able to load a .tiff file ok. When I click "run ROI Detection" I get the following

Undefined function 'strel' for input arguments of type 'char'.

Error in CNMFSetParms (line 301)
{strel('disk',1,0)}

Error in ez_roi_detect/RunROIDetectionButtonPushed (line 345)
options = CNMFSetParms(... % Check CNMFSetParms.m for more details

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 386)
Error while evaluating Button PrivateButtonPushedFcn.

running on R2020b, Mac OS 11.1

cheers
Leo

Hi, I have read the paper and it seems like a great toolbox.
Wanted to try but have some trouble. I have Matlab 2018a for MacOS, I have all 4 required packaged installed and installed (with path and all) EZcalcium, CaImAn, and NoRMCorre.
Still there is something I am missing. the command ezcalcium opens the first GUI, but then only the help button works, all others give me some trouble with 'getRunningApp'. Below is the error. Any idea?

cheers,

Marco

**Undefined function 'getRunningApp' for input arguments of type
'ez_motion_correction'.

Error in ez_motion_correction (line 663)
runningApp = getRunningApp(app);

Error in ezcalcium/MotionCorrectionButtonPushed (line 22)
ez_motion_correction

Error using matlab.ui.control.internal.controller.ComponentController/executeUserCallback (line 309)
Error while evaluating Button PrivateButtonPushedFcn.**

Hi, I am trying to use EZCalcium to identify when signal in my tissue of interest is lost. When I compare across multiple files, each corresponding to time points before and after signal is lost, the ROI matching will produce an empty set of overlapping ROIs, since the "ROIs" that EZCalcium detects when no signal is present are not meaningful.

Is there a way to extract the positional information (i.e., centers and borders) of each ROI for a given file? This way I can do the delta F / F analysis on a given region in ImageJ.

Hi!

I'm getting the following error during the generation of the motion corrected tiff stack.

Warning: TIFF library warning - 'TIFFReadDirectory: Unknown field with tag 50838 (0xc696) encountered.'
Warning: TIFF library warning - 'TIFFReadDirectory: Unknown field with tag 50839 (0xc697) encountered.'

The resulting file does not open/appears corrupted. Advice would be much appreciated!

Hello,

I'm having some issues exporting my data as a .mat file after completing the ROI Refinement. I run through all the ROI and once I click 'export data' the system crashes and the entire Matlab app closes out. This is true for all my files, even on files that I have exported in the past.

I've tried restarting everything (computer and program), uninstalling and installing again as well as using the newest version of MATLAB 2022a. Still no luck. Other Matlab features seem to work fine so now I'm assuming it is an EZCalcium issue.

I'd appreciate any help. Thanks!