/// <summary>
/// Return copy of the curve filtered according to the filter settings (shorter by double the filter size)
/// </summary>
public double[] GetFilteredYs(double[] curve)
{
if (curve == null)
{
return(null);
}
double[] filteredValues = ImageDataTools.GaussianFilter1d(curve, filterPx);
int padPoints = filterPx * 2 + 1;
double[] filteredYs = new double[curve.Length - 2 * padPoints];
Array.Copy(filteredValues, padPoints, filteredYs, 0, filteredYs.Length);
return(filteredYs);
}
/// <summary>
/// Set the structure bounds around the brightest structure in the image.
/// The 1D projected image of the green channel is used.
/// Boundaries are placed where intensity drops to half the distance between the peak and the noise floor.
/// Noise floor is defined as the 20-percentile of the 1D projected image.
/// </summary>
public void AutoStructure()
{
// determine the brightest column
double[] columnIntensities = ImageDataTools.GetAverageLeftright(imgG);
double brightestValue = 0;
int brightestIndex = 0;
for (int i = 0; i < columnIntensities.Length; i++)
{
if (columnIntensities[i] > brightestValue)
{
brightestValue = columnIntensities[i];
brightestIndex = i;
}
}
Log($"Brightest structure (G): {brightestValue} AFU at {brightestIndex}px");
// determine the 20-percentile brightness (background fluorescence)
double[] sortedIntensities = new double[columnIntensities.Length];
Array.Copy(columnIntensities, sortedIntensities, columnIntensities.Length);
Array.Sort(sortedIntensities);
double noiseFloor = sortedIntensities[(int)(sortedIntensities.Length * .2)];
Log($"Noise floor (G): {noiseFloor} AFU");
// intensity cut-off is half-way to the noise floor
double peakAboveNoise = brightestValue - noiseFloor;
double cutOff = (peakAboveNoise * .5) + noiseFloor;
Log($"Cut-off (G): {cutOff} AFU");
// start both structures at the brighest point, then walk away
structure1 = brightestIndex;
structure2 = brightestIndex;
while (columnIntensities[structure1] > cutOff && structure1 > 0)
{
structure1--;
}
while (columnIntensities[structure2] > cutOff && structure2 < columnIntensities.Length - 1)
{
structure2++;
}
Log($"Automatic structure: {structure1}px - {structure2}px");
}
/// <summary>
/// This is the primary analysis method for delta green and delta green over red calculations
/// </summary>
public void GenerateAnalysisCurves()
{
// determine mean pixel intensity over time between the structure markers
var(structureIndex1, structureIndex2) = GetValidStructure(structure1, structure2);
curveG = ImageDataTools.GetAverageTopdown(imgG, structureIndex1, structureIndex2);
curveR = ImageDataTools.GetAverageTopdown(imgR, structureIndex1, structureIndex2);
// create a dG channel by baseline subtracting just the green channel
var(baselineIndex1, baselineIndex2) = GetValidBaseline(baseline1, baseline2);
curveDeltaG = CreateBaselineSubtractedCurve(curveG, baselineIndex1, baselineIndex2);
// create a d(G/R) curve by finding the G/R ratio then baseline subtracting that
if (isRatiometric)
{
curveGoR = CreateRatioCurve(curveG, curveR);
curveDeltaGoR = CreateBaselineSubtractedCurve(curveGoR, baselineIndex1, baselineIndex2);
}
}
/// <summary>
/// Multi-scan linescans have multiple frames.
/// Call this to load data for a specific frame.
/// </summary>
public void SetFrame(int frameNumber)
{
// error checking
if (!isValid)
{
return;
}
int frameCount = Math.Max(pathsDataG.Length, pathsDataR.Length);
if (frameNumber >= frameCount)
{
Log($"frame {frameNumber} cannot be called (max frame {frameCount - 1})");
return;
}
// load data bitmaps and create ratio if needed
if (pathsDataG.Length > 0)
{
imgG = new ImageData(pathsDataG[frameNumber]);
bmpDataG = imgG.GetBmpDisplay();
}
if (pathsDataR.Length > 0)
{
imgR = new ImageData(pathsDataR[frameNumber]);
bmpDataR = imgR.GetBmpDisplay();
}
if (bmpDataG != null && bmpDataR != null)
{
bmpDataM = ImageDataTools.Merge(imgR, imgG, imgR);
double[] dataM = new double[imgG.data.Length];
for (int i = 0; i < dataM.Length; i++)
{
dataM[i] = imgG.data[i] / imgR.data[i];
}
imgM = new ImageData(dataM, imgG.width, imgG.height);
}
}
public void UpdateGraphs(bool skipUpdatesIfBusy = true)
{
// use pretty colors
Color blue = System.Drawing.ColorTranslator.FromHtml("#1f77b4");
Color lightBlue = System.Drawing.ColorTranslator.FromHtml("#a2d1f2");
Color red = System.Drawing.ColorTranslator.FromHtml("#d62728");
Color lightRed = System.Drawing.ColorTranslator.FromHtml("#e63738");
Color green = System.Drawing.ColorTranslator.FromHtml("#2ca02c");
Color lightGreen = System.Drawing.ColorTranslator.FromHtml("#98df8a");
Color colorBaselineMarks = System.Drawing.ColorTranslator.FromHtml("#666666");
Color colorPeak = System.Drawing.ColorTranslator.FromHtml("#d62728");
Color colorZero = System.Drawing.ColorTranslator.FromHtml("#000000");
if (!lsFolder.isValid)
{
return;
}
if (skipUpdatesIfBusy && busyUpdating)
{
return;
}
else
{
busyUpdating = true;
}
// do the analysis
lsFolder.GenerateAnalysisCurves();
// update the structure profile plot
double[] columnPixelIndices = new double[lsFolder.imgG.width];
for (int i = 0; i < columnPixelIndices.Length; i++)
{
columnPixelIndices[i] = i;
}
formsPlot2.plt.Clear();
if ((cbR.Enabled && cbR.Checked))
{
double[] columnBrightnessR = ImageDataTools.GetAverageLeftright(lsFolder.imgR);
formsPlot2.plt.PlotScatter(columnPixelIndices, columnBrightnessR, markerSize: 0, color: red);
}
if ((cbG.Enabled && cbG.Checked))
{
double[] columnBrightnessG = ImageDataTools.GetAverageLeftright(lsFolder.imgG);
formsPlot2.plt.PlotScatter(columnPixelIndices, columnBrightnessG, markerSize: 0, color: green);
}
formsPlot2.plt.AxisAuto(0, .1);
formsPlot2.plt.PlotVLine(lsFolder.structure1, color: Color.Black);
formsPlot2.plt.PlotVLine(lsFolder.structure2, color: Color.Black);
// update styling before the render
formsPlot2.plt.YLabel("Intensity");
formsPlot2.plt.XLabel("Position (pixel number)");
formsPlot2.Render();
// update the time series plot
curveToCopy = null;
formsPlot1.plt.Clear();
if (cbRatio.Checked && cbRatio.Enabled)
{
if (cbDelta.Enabled && cbDelta.Checked)
{
curveToCopy = lsFolder.GetFilteredYs(lsFolder.curveDeltaGoR);
formsPlot1.plt.PlotScatter(lsFolder.timesMsec, lsFolder.curveDeltaGoR, color: lightBlue, lineWidth: 0, markerSize: 2);
formsPlot1.plt.PlotScatter(lsFolder.GetFilteredXs(), curveToCopy, markerSize: 0, color: blue, lineWidth: 2);
formsPlot1.plt.PlotVLine(lsFolder.baseline1 * lsFolder.scanLinePeriod, color: colorBaselineMarks, lineWidth: 2);
formsPlot1.plt.PlotVLine(lsFolder.baseline2 * lsFolder.scanLinePeriod, color: colorBaselineMarks, lineWidth: 2);
formsPlot1.plt.PlotHLine(0, color: colorZero, lineWidth: 2);
formsPlot1.plt.PlotHLine(curveToCopy.Max(), color: colorPeak, lineWidth: 2);
formsPlot1.plt.YLabel("Delta G/R (%)");
}
else
{
curveToCopy = lsFolder.GetFilteredYs(lsFolder.curveGoR);
formsPlot1.plt.PlotScatter(lsFolder.timesMsec, lsFolder.curveGoR, color: lightBlue, lineWidth: 0, markerSize: 2);
formsPlot1.plt.PlotScatter(lsFolder.GetFilteredXs(), curveToCopy, markerSize: 0, color: blue, lineWidth: 2);
formsPlot1.plt.YLabel("G/R (%)");
formsPlot1.plt.PlotHLine(curveToCopy.Max(), color: colorPeak, lineWidth: 2);
}
}
else if ((cbR.Checked && cbR.Enabled) && (cbG.Checked && cbG.Enabled))
{
formsPlot1.plt.PlotScatter(lsFolder.timesMsec, lsFolder.curveG, markerSize: 0, color: green);
formsPlot1.plt.PlotScatter(lsFolder.timesMsec, lsFolder.curveR, markerSize: 0, color: red);
formsPlot1.plt.YLabel("G and R (AFU)");
}
else if (cbR.Checked && cbR.Enabled)
{
formsPlot1.plt.PlotScatter(lsFolder.timesMsec, lsFolder.curveR, markerSize: 0, color: red);
formsPlot1.plt.YLabel("R (AFU)");
}
else if (cbG.Checked && cbG.Enabled)
{
if (cbDelta.Enabled && cbDelta.Checked)
{
curveToCopy = lsFolder.GetFilteredYs(lsFolder.curveDeltaG);
formsPlot1.plt.PlotScatter(lsFolder.timesMsec, lsFolder.curveDeltaG, lineWidth: 0, color: lightGreen, markerSize: 2);
formsPlot1.plt.PlotScatter(lsFolder.GetFilteredXs(), curveToCopy, markerSize: 0, color: green, lineWidth: 2);
formsPlot1.plt.PlotVLine(lsFolder.baseline1 * lsFolder.scanLinePeriod, color: colorBaselineMarks, lineWidth: 2);
formsPlot1.plt.PlotVLine(lsFolder.baseline2 * lsFolder.scanLinePeriod, color: colorBaselineMarks, lineWidth: 2);
formsPlot1.plt.PlotHLine(0, color: colorZero, lineWidth: 2);
formsPlot1.plt.PlotHLine(curveToCopy.Max(), color: colorPeak, lineWidth: 2);
formsPlot1.plt.YLabel("Delta G (%)");
}
else
{
curveToCopy = lsFolder.GetFilteredYs(lsFolder.curveG);
formsPlot1.plt.PlotScatter(lsFolder.timesMsec, lsFolder.curveG, lineWidth: 0, color: lightGreen, markerSize: 2);
formsPlot1.plt.PlotScatter(lsFolder.GetFilteredXs(), curveToCopy, markerSize: 0, color: green, lineWidth: 2);
formsPlot1.plt.PlotHLine(curveToCopy.Max(), color: colorPeak, lineWidth: 2);
formsPlot1.plt.YLabel("G (AFU)");
}
}
formsPlot1.plt.AxisAuto(.05, .1);
// make ticks smaller than typical
var tickFont = new Font(FontFamily.GenericMonospace, (float)10.0);
//formsPlot1.plt.Ticks(font: tickFont);
//formsPlot2.plt.Ticks(font: tickFont);
// update styling before the render
formsPlot1.plt.XLabel("Time (milliseconds)");
formsPlot1.Render();
// update peak label
if (curveToCopy == null)
{
lblPeak.Text = $"";
}
else
{
lblPeak.Text = $"{Math.Round(curveToCopy.Max(), 2)}%";
}
Application.DoEvents();
busyUpdating = false;
}
