# A Plug-in Server

Scanbox has a memory mapped mechanism to share the incoming data stream with other processes that intend to consume the data in real-time.

Examples of simple uses of such data sharing include the calculation of rolling averages, displaying a real-time histogram, or generating a montage display for volumetric data.

Of course, different experiments may need to be processed by different plug-ins.  To simplify this process we now provide a single mechanism that allows the selection of a plug-in via a pull-down menu in the Scanbox GUI.

The configuration variable “plugin” can be used to list a number of different options to call.  In the example below, two possible options are listed.

sbconfig.plugin = {'rolling','montage'}; %plugin options


These must be names of Matlab scripts that will be called to process the data.

When Scanbox starts it will list these options in a pull down menu within the real-time processing panel, along with two check-boxes, one labeled “plugin” and another labeled “focus”.

The pull down menu allows the user to select one plugin among the available selections.

The plug-in checkbox indicates you want the data to be shared via the memory mapped mechanism, and the “focus” checkbox indicates whether you want the data to be shared not only during a grabbing operation, but also when you are focusing.

For example, adjusting the laser intensity by analyzing the histogram of values in the incoming data may be something you want to do ahead of an actual experiment, and something you will want to run while focusing.

Now, when you start Scanbox a new Matlab instance is opened and calls the sbxplugin_server.m script (assuming mmap=true in the config file).  This is a simple script that waits for new data to arrive and calls the selected plug-in.

% Scanbox plug in server script
% This code will run upon startup of Scanbox on a separate matlab instance if mmap=true
% in the scanbox_config file

% Open memory mapped file -- define just the header first

scanbox_config; % read the scanbox config structure

mmfile = memmapfile('scanbox.mmap','Writable',true, ...
'Format', { 'int16' [1 16] 'header' } , 'Repeat', 1);
flag = 1;

% Process all incoming frames until Scanbox stops

running = false;

while(true)

disp('Waiting for imaging data stream');

while ~running
end

plugin_id = mmfile.Data.header(7); % get the plug in to be used

fprintf('Plugin id=%d\n',plugin_id);

while running

running = (mmfile.Data.header(1) ~= -2); % no stop signal present

if running && mmfile.Data.header(1)>=0 % if not stopped and frame present

switch plugin_id

case 1
% replace the line below with your own script for
% plugin with id #1

case 2
% replace the line below with your own script for
% plugin with id #2

otherwise
disp('Invalid plugin id number');
end

mmfile.Data.header(1) = -1; % signal Scanbox that frame has been consumed!

end
end
end

clear(mmfile); % close the memory mapped file
close all; % close all figures


The default server code simply lists number of of the incoming frame and the selected plug-in.  Just add an eval() call to the script name at the indicated locations.

The output of the script, as is, will look like:


Waiting for imaging data stream
Plugin id=2
ROI=0 Expt=0
Frame: 00000 Plugin: montage
Frame: 00001 Plugin: montage
Frame: 00002 Plugin: montage
Frame: 00003 Plugin: montage
Frame: 00004 Plugin: montage
Frame: 00005 Plugin: montage
Frame: 00006 Plugin: montage
Frame: 00007 Plugin: montage
Frame: 00008 Plugin: montage
Frame: 00009 Plugin: montage
Frame: 00010 Plugin: montage
Waiting for imaging data stream



The user can now easily switch between plugins from the Scanbox window by changing the selection in the pull down menu.

# New alignment and segmentation tools

Improved alignment and segmentation tools have now been released in the latest version of Scanbox, while retaining much of the functionality of the last version.

sbxaligntool. The new alignment tool, shown below, adds batch processing of files, including the processing of eye and ball motion if those data are present.  A region-of-interest (ROI) can optionally be selected manually or automatically.  For file entries where manual selection was specified, the program will stop and present a rectangle on the screen for the user to specify the ROI.  Typically, automatic ROI works fine, and it does not require the user to stand by the computer to specify the ROI each time a new file starts to process.

As the files are aligned, the Status column and Status message will display the progress. The alignment procedure can also be visualized by clicking the Live update checkbox, which will display the mean of the entire image stack as the process moves along.  Pan and Zoom buttons allow the user to inspect details in the live image, such as fine branches, as the system is carrying out the alignment. This tool performs rigid alignment and the result is stored in a *_rigid.sbx file.  The original data is left untouched. The tool can align images relatively fast (about 65 frames/sec in my computer), but it will take a few minutes to compute the reference image if the sequence is 15 min or more (please be patient). Alignment improves with the number of passes requested.  Usually one pass is very good, but you can try two or more passes by changing the appropriate entry in the column. The alignment algorithm has been improved.

sbxsegmenttool. The segmentation tool works in a similar way as before. After loading the aligned *_rigid.sbx file, it will display the correlation map.  Segmentation then proceeds as in the previous version.

Once a number of cells are selected, you must save the segmentation and then extract the signals by pressing the corresponding buttons. After the signals are extracted you can select a cell with the pull down menu on the bottom left and the traces corresponding to that cell (now highlighted in green) will be displayed.  The blue trace represents the average signal within the cell, the gray trace is the neuropil, and the trace is the estimated spike rate using the Vanilla algorithm with parameters optimized for GCaMP6f.

Improvements include an Undo button, which will remove the last cell segmented. The ability to load a previous segmentation (it will load automatically after you select the *_rigid.sbx file), to continue adding cells to it.  The ability to define a ROI in the correlation map to automatically increase the contrast of the correlation map as the most salient cells are selected. A zoomed version of the mean image on the right to go along with the correlation map.  And the tool now saves the neuropil and deconvolved signal as well.

Give these tools a try. Report back any suggestions for improvements or problems you encounter.

# Spikefinder: the Vanilla algorithm

Over the last few months, the Spikefinder challenge has provided a playing ground for colleagues to offer ideas about how to estimate the spiking of individual neurons based on the measured activity of fluorescent calcium indicators.

The challenge was for people to come up with strategies that beat the performance of state-of-the-art algorithms, STM & oopsi. A good number of algorithms were able to achieve this in short time, including one I submitted, termed Vanilla.

The best performing algorithms seem to have relied on modern machine learning methods. Vanilla is nothing more than a linear filter followed by a static-nonlinearity $y(t) = \phi ( h(t) * x(t) )$ — thus the name.

The filter $h(t)$ is a linear combination of an even filter, estimating the mean of the signal at time $t$, and an odd filter, estimating the derivative of the signal at time $t$.

The even filter is a Gaussian, $h_{even} = A \exp ( -t^2 / 2 \sigma^2 )$, and the odd filter is the derivative of a Gaussian $h_{odd} = B t \exp (-t^2 / 2 \sigma^2)$.  The constants $A$ and $B$ are such that the norm of the filters is normalized to one, $\| A \| = \| B \| = 1$.  These two filters are linearly combined while keeping the norm of resulting filter equal to one, $h(t) = \cos \alpha \: h_{even}(t) + \sin \alpha \: h_{odd}(t)$.

The output nonlinearity is a rectifier to a power, $\phi ( x ) = (x- \theta)^\beta$ if $x>\theta$, and zero otherwise.

The model has only 4 parameters, $\{\sigma, \alpha, \theta, \beta \}$. The amount of smoothing of the signal is controlled by $\sigma$, the shape of the filter is controlled by $\alpha$, and the threshold $\theta$ and power $\beta$ determine the shape of the nonlinearity.

The model is fit by finding the optimal values of $\{\sigma, \alpha, \theta, \beta \}$ that maximize the correlation between its output $y(t)$ and the recorded spiking of the neuron.  I used Matlab’s fminsearch() to perform this optimization, which was typically finished in about 60 sec or less for most datasets.

The only pre-processing done was a z-scoring of the raw signals.  In one dataset (dataset #5, GCaMP6s in V1), we allowed for an extra-delay parameter between the signal and the prediction.

I was surprised this entry did relatively well, as the algorithm it is basically a version of STM. I think the particular shape of the output nonlinearity (rectifier+power vs exponential), the constrain imposed on the shape of the filters, and the resulting small number of parameters, paid a role in Vanilla doing better overall.

The top algorithms reached an absolute performance of about 0.47 and it seems unlikely this performance can be improved by a lot. This seems to highlight the limitations of the current families of calcium indicators in yielding precise spiking information — so there is plenty of opportunity to improve them.

It is interesting that despite its simplicity, the relative performance of Vanilla, with a correlation coefficient of 0.428 was not dramatically inferior to that of the top performing, deep network models, with all its bells and whistles, which landed at 0.464.  So, one must pay due respects to deep networks, but I was honestly expecting Vanilla to be completely blown out of the water in terms of performance, and I don’t think it was.

Finally, Vanilla is a non-casual algorithm, as both past and future samples are used to predict the response at time $t$. In some situations, however, when we are trying close the loop as fast as possible by controlling a stimulus based on neural activity itself, we need algorithms that are casual and can provide a fast, online estimate of spiking activity. I wonder if any of the submissions are causal algorithms and, if not, what would be the best performance the methods can attain if we allow them to provide estimates of spiking based only on past samples.

# External TTL trigger

To automatically start /stop acquisition by means of an external TTL signal follow the following instructions.

First, connect an appropriate TTL control signal to the P1.6 (pin #21) of the extension header of the Scanbox board.  The view below shows a top view of the Scanbox control board.  The pin in question is located on the back row of connectors when viewing the board from the front.  As a ground pin, you can use pin #3.  To make it easier to make the appropriate connections it helps to get the this cable and route it outside the box.

Start the Scanbox software and operate as usual by focusing and selecting the area you want to record.  When ready to switch to external trigger control, simply click the “External TTL Trigger” checkbox, which is located in the middle of the Scanner control panel.

After enabling the TTL trigger, the manual Focus/Grab buttons will be grayed out and blocked from usage.  If you want to go back to manual control simply deselect the TTL Trigger checkbox.

The rising edge of the TTL control signal is used to start/stop the microscope.  Minimum pulse width is 1 ms.

While controlling the microscope using an external TTL signal it is useful to run it in continuous resonant mode (so you avoid waiting for the resonant mirror to warm up) , and set the “autoinc” configuration variable to “true”, so file numbers increment automatically after the completion of each session.

To use this feature you have to update to the latest version of the firmware/software.

Update [6/23/17]:

If you are willing to give use of TTL1 as an event line, a new trig_sel configuration variable allows you to use it instead of the signal from the header to start and stop the acquisition.  You will have to upgrade to the latest version of the software.  Follow the instructions here.  You can skip the steps asking you to update knobby, the motor box, and run vc_redist.x64.exe.  After the update, simple set the trig_sel configuration variable to true if you want to trigger with TTL1 (or false if you want to trigger using the header signal).