Ephic (Electrophysiology and Imaging with C++)

Tutorial #22

Processing of Imaging Data

Let's consider this example. We have one baseline record and two bAP records. Each bAP record contains two bAPs separated by 200 ms. We want to measure the peak amplitude of calcium imaging signals evoked by individual bAPs. We first load the baseline record and click "Save Slope". Check "Bleach Correction" and "Data Inversion". We check "Temporal Filter" with "Half Width = 1" to reduce noise.
The voltage trace in the bAP record is displayed on the right. Create ROIs along the dendrite. The corresponding traces are displayed in the imaging trace window. The blue trace in the imaging trace window indicates the interval between two consecutive points. The height is one unit from the beginning to the end, indicating the interval is 1 ms from the beginning to the end without missing any frame. You can hide this trace by unchecking the "Interval" box. The number at the top left corner is the upper boundary of the window.
Average Two Records Click "Add" button in the "Averaging" panel. Go to the next record and click "Add" button again. You will see the "NumRec" box display "2", indicating there are two records in the buffer. Check "Active" and the imaging window and the imaging trace window will display the averaged data. You might notice the noise is lower. To clear the data in the buffer, click "Clear" button. The number in the "NumRec" box will become "0" and the "Active" box will be unchecked automatically.
To remove baseline, check "Baseline Removal". To translate the baseline to the middle of the window, check "Centered".	Baseline Removed	Centered
To divide the data by baseline intensity, check "Data Divided by F0". To adjust the y scale, slide the "Y Scale" slider. Now, the upper boundary is 14.48%. You can adjust the x scale by sliding the "X Scale" slider. You can also translate the trace along the x axis by sliding the "X Shift" slider. You can change the background color to white by clicking "Background" and select "White". You can save the imaging trace window to a TGA file by clicking "Save Image" and following the instructions. You can save the traces to a plain text file by clicking "Save Traces" and following the instructions.	Data Divided by F0	Y Scale Adjusted
Ephic measures the average, the standard deviation, and the peak amplitude of the whole trace of the active ROI and displays the values in the "Analysis of Signals in ROIs" panel. To measure these characteristics in a specified period, check "On" box, type in the start point and the width in the "Start" and the "Width" boxes, and press "Enter". You will see two orange vertical lines indicating the boundaries of this analysis window.	Characteristics of The Active ROI	Analysis Window
To save the peak value of each trace, click "Save Max" and the values will be saved to "Max.txt". To subtract RMS value from the max, check "RMS Removal" box. The RMS value is calculated in the period defined in the "Data Processing" tab.		"Max.txt" No Max 0 11.2013 1 6.5584 2 5.56005 3 4.8601 4 4.183 5 3.751
To save the peak values of both calcium transients, type in "90", "200", "2" in "Start", "Width", and "Numver" boxes and press "Enter". Click "Save Maxima" button and the values will be saved to "Maxima.txt".		"Maxima.txt" No Max1 Max2 0 11.2013 11.8885 1 6.5584 7.38292 2 5.56005 7.11527 3 4.8601 5.44764 4 4.183 4.79142 5 3.751 3.58588
Background Subtraction Create background ROIs. Click "Save BG" to save the minimum F0 in the box to a background buffer. Load original ROIs and check "Background Subtraction".	Background ROIs