{

INCLUSION COUNTER: Batch Count Inclusions v1.0
Jeremy Brown 1997


---------------------------------------------------------

You must have run the macro "Help With Paths" when you first installed Inclusion counter on this Mac or these macros WILL NOT RUN!

If you did not, DO SO NOW!

This macro-file must be loaded into NIH Image to run.

To Load "Batch Count Inclusions v1.0" choose "Load Macros From Window" from the "Special" menu then close this window. Alternatively press Apple-9 followed by Apple-W.

The range of macros that constitute this part of Inclusion Counter will then be displayed in the "Special" menu.

---------------------------------------------------------





}

Var
left,top,width,height,ImageScale,MaxArea,TotalMaxArea,MinArea,TotalMinArea, MeanValue, n, m, radius, q, i, lower, upper, min, max, x1,x2,y1,y2,ignore1:integer;
Sum, SumOfSquares:Boolean;
ImageUnits:String;

procedure ApplyROI;
begin
     Open('PASTEHERE');
end;
end;

procedure CheckForStack;
begin
    if nPics=0 then begin
    PutMessage('This macro requires a stack! Please open your Infected and Uninfected control Images and choose "Windows to Stack" from the Stacks menu.');
    exit;
end;
    if nSlices=0 then begin
    PutMessage('This window is not a stack! Please open your Infected and Uninfected control Images and choose "Windows to Stack" from the Stacks menu.');
    exit
end;
end;

procedure CheckStack;
begin
    if nPics=0 then begin
    PutMessage('This macro requires a RGB stack! Please open a typical example image of infected cells.');
    exit;
end;
    if nSlices=0 then begin
    PutMessage('This window is not a stack! Please cheack the format of the images, see the mannual for details.');
    exit;
end;
end;

Procedure MaxMin;
begin
   SetOptions('Area');         {may want to add other options}
   SetParticleSize(1,10000);  {as desired}
   MinArea:=1;
   MaxArea:=10000;
for i:=1 to rcount do begin
if i=1 then begin
   MinArea:=rArea[i];
   MaxArea:=rArea[i];
end else begin
if rArea[i]>MaxArea then MaxArea:=rArea[i];
if rArea[i]<MinArea then MinArea:=rArea[i];
end;
if i=rCount then TotalMaxArea:=MaxArea;
if i=rCount then TotalMinArea:=MinArea;
end;
  SelectWindow('Controls');
  Write('                     ', TotalMaxArea);
  WriteLn('                             ', TotalMinArea);
end;
end;

Procedure MaxMin2;
begin
   SetOptions('Area');         {may want to add other options}
   SetParticleSize(1,10000);  {as desired}
   MinArea:=1;
   MaxArea:=10000;
for i:=1 to rcount do begin
if i=1 then begin
   MinArea:=rArea[i];
   MaxArea:=rArea[i];
end else begin
if rArea[i]>MaxArea then MaxArea:=rArea[i];
if rArea[i]<MinArea then MinArea:=rArea[i];
end;
if i=rCount then TotalMaxArea:=MaxArea;
if i=rCount then TotalMinArea:=MinArea;
end;
  SelectWindow('Controls II');
  Write('                  ', TotalMaxArea);
  Write('                          ', TotalMinArea);
end;
end;

procedure MeanAreaValue;
begin
      Sum := 0.0;
      For i := 1 to rCount do begin
      Sum := Sum + rArea[i];
end;
Write('                   ',Sum/rCount:0:0);
end;
end;

Procedure StandardDeviation;
begin
If (rCount<3) then Begin;
   WriteLn ('                     0');
end Else begin;
   SumOfSquares := 0.0;
   For i := 1 to rCount do begin;
   MeanValue := Sum/rCount;
   SumOfSquares:= SumOfSquares + Sqr( rArea[i] - MeanValue);
end;
   n := rcount-1;
   M := SumOfSquares / n;
   WriteLn ('                   ',sqrt(M));
end;
end;

Procedure DeleteTouching;
begin
   SetThreshold(1);
   MakeBinary;
   WandAutoMeasure(false);
   GetROI(left,top,width,height);
   MakeLineROI(left,top,left+width-1,top);
   Drawboundary;
   AutoOutline(left,top);
   Clear;
   SetThreshold(1);
   MakeBinary;
   MakeLineROI(left,top,left,top+Height-1);
   Drawboundary;
   AutoOutline(left,top);
   Clear;
   SetThreshold(1);
   MakeBinary;
   MakeLineROI(left+width-1,top,left+width-1,top+Height-1);
   Drawboundary;
   AutoOutline(left+width-1,top);
   Clear;
   SetThreshold(1);
   MakeBinary;
   MakeLineROI(left,top+Height-1,left+width-1,top+Height-1);
   Drawboundary;
   AutoOutline(left,top+Height-1);
   Clear;
   SetThreshold(1);
   MakeBinary;
end;
end;

procedure ROISize;
begin
     PutMessage('Please open your first image.');
     Open('');
     ApplyROI;
     SetScale(ImageScale,' ImageUnits');
     ResetCounter;
     Measure;
     n:= rArea[1];
     Dispose;
     SelectWindow ('Inclusions');
     WriteLN('          ROI Area ='  n, ImageUnits);
end
end

Procedure Red;
begin
    SelectSlice(3);
    DeleteSlice;
    SelectSlice(2);
    DeleteSlice;
end;
End;

Procedure Green;
begin
    SelectSlice(3);
    DeleteSlice;
    SelectSlice(1);
    DeleteSlice;
end;
End;

Procedure Blue;
begin
    SelectSlice(2);
    DeleteSlice;
    SelectSlice(1);
    DeleteSlice;
end;
End;

Procedure RedGreen2;
begin
    SelectSlice(3);
    DeleteSlice;
    AverageSlices;
    SelectWindow('Example Image');
    Dispose;
    SelectWindow('Average');
end;
End;

Procedure RedGreen;
begin
    SelectSlice(3);
    DeleteSlice;
    AverageSlices;
    SelectWindow('Average');
end;
End;

Procedure RedBlue2;
begin
    SelectSlice(2);
    DeleteSlice;
    AverageSlices;
    SelectWindow('Example Image');
    Dispose;
    SelectWindow('Average');
end;
End;

Procedure RedBlue;
begin
    SelectSlice(2);
    DeleteSlice;
    AverageSlices;
    SelectWindow('Average');
end;
End;

Procedure GreenBlue2;
begin
    SelectSlice(1);
    DeleteSlice;
    AverageSlices;
    SelectWindow('Example Image');
    Dispose;
    SelectWindow('Average');
end;
End;

Procedure GreenBlue;
begin
    SelectSlice(1);
    DeleteSlice;
    AverageSlices;
    SelectWindow('Average');
end;
End;

Macro '[A] About Batch Count Inclusions v1.0'
begin
      PutMessage('These macros are designed to take you step by step through the process of batch processing images of inclusions.');
end;
end;

Macro '[C] Choose a Colour Channel';
begin 
    CheckStack;
    SetSaveAS('Tiff');
    SaveAs('Example Image');
    Dispose;
    Open('Example Image');
    SubtractBackground('2D Rolling Ball',100);
    Smooth;
    SelectSlice(2);
    SubtractBackground('2D Rolling Ball',100);
    Smooth;
    SelectSlice(3);
    SubtractBackground('2D Rolling Ball',100);
    Smooth;
    SaveAs('Example Image');
     SetPlotSize(510,255);
     SetPlotScale(1,255);
     SetNewSize(700,572);
     MakeNewStack('Channels Stack');
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      AddSlice;
      Open('Example Image');
      AverageSlices;
      SelectAll;
      Copy;
      SelectWindow('Channels Stack');
      ChooseSlice(1);
      Paste;
      SurfacePlot;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(8);
      Paste;
      SelectWindow('Average');
      SelectAll;
      GetROI(left,top,width,height);
      MakeLineROI(left,top,left+width-1,top+height-1);
      PlotProfile;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(15);
      Paste;
      SelectSlice(1);
      MoveTo(15,15);
      SetFontSize(14);
      SetFont('Helvetica');
      SetText('Bold');
      Write(' GREY SCALE');
      SelectSlice(8);
      MoveTo(15,15);
      Write(' GREY SCALE');
      SelectSlice(15);
      MoveTo(15,15);
      Write(' GREY SCALE');
      SelectWindow('Average');
      Dispose;
      Open('Example Image');
      Red;
      SelectAll;
      Copy;
      SelectWindow('Channels Stack');
      ChooseSlice(2);
      Paste;
      SurfacePlot;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(9);
      Paste;
      SelectWindow('Example Image');
      SelectAll;
      GetROI(left,top,width,height);
      MakeLineROI(left,top,left+width-1,top+height-1);
      PlotProfile;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(16);
      Paste;
      SelectSlice(2);
      MoveTo(15,15);
      SetFontSize(14);
      SetFont('Helvetica');
      SetText('Bold');
      Write(' RED CHANNEL');
      SelectSlice(9);
      MoveTo(15,15);
      Write(' RED CHANNEL');
      SelectSlice(16);
      MoveTo(15,15);
      Write(' RED CHANNEL');
      SelectWindow('Example Image');
      Dispose;
      Open('Example Image');
      Green;
      SelectAll;
      Copy;
      SelectWindow('Channels Stack');
      ChooseSlice(3);
      Paste;
      SurfacePlot;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(10);
      Paste;
      SelectWindow('Example Image');
      SelectAll;
      GetROI(left,top,width,height);
      MakeLineROI(left,top,left+width-1,top+height-1);
      PlotProfile;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(17);
      Paste;
      SelectSlice(3);
      MoveTo(15,15);
      Write(' GREEN CHANNEL');
      SelectSlice(10);
      MoveTo(15,15);
      Write(' GREEN CHANNEL');
      SelectSlice(17);
      MoveTo(15,15);
      Write(' GREEN CHANNEL');
      SelectWindow('Example Image');
      Dispose;
      Open('Example Image');
      Blue;
      SelectAll;
      Copy;
      SelectWindow('Channels Stack');
      ChooseSlice(4);
      Paste;
      SurfacePlot;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(11);
      Paste;
      SelectWindow('Example Image');
      SelectAll;
      GetROI(left,top,width,height);
      MakeLineROI(left,top,left+width-1,top+height-1);
      PlotProfile;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(18);
      Paste;
      SelectSlice(4);
      MoveTo(15,15);
      Write(' BLUE CHANNEL');
      SelectSlice(11);
      MoveTo(15,15);
      Write(' BLUE CHANNEL');
      SelectSlice(18);
      MoveTo(15,15);
      Write(' BLUE CHANNEL');
      SelectWindow('Example Image');
      Dispose;
      Open('Example Image');
      RedGreen2;
      SelectAll;
      Copy;
      SelectWindow('Channels Stack');
      ChooseSlice(5);
      Paste;
      SurfacePlot;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(12);
      Paste;
      SelectWindow('Average');
      SelectAll;
      GetROI(left,top,width,height);
      MakeLineROI(left,top,left+width-1,top+height-1);
      PlotProfile;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(19);
      Paste;
      SelectSlice(5);
      MoveTo(15,15);
      Write('  RED & GREEN CHANNELS');
      SelectSlice(12);
      MoveTo(15,15);
      Write('  RED & GREEN CHANNELS');
     SelectSlice(19);
      MoveTo(15,15);
      Write('  RED & GREEN CHANNELS');
      SelectWindow('Average');
      Dispose;
      Open('Example Image');
      RedBlue2;
      SelectAll;
      Copy;
      SelectWindow('Channels Stack');
      ChooseSlice(6);
      Paste;
      SurfacePlot;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(13);
      Paste;
      SelectWindow('Average');
      SelectAll;
      GetROI(left,top,width,height);
      MakeLineROI(left,top,left+width-1,top+height-1);
      PlotProfile;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(20);
      Paste;
      SelectSlice(6);
      MoveTo(15,15);
      Write('  RED & BLUE CHANNELS');
      SelectSlice(13);
      MoveTo(15,15);
      Write('  RED & BLUE CHANNELS');
      SelectSlice(20);
      MoveTo(15,15);
      Write('  RED & BLUE CHANNELS');
      SelectWindow('Average');
      Dispose;
      Open('Example Image');
      GreenBlue2;
      SelectAll;
      Copy;
      SelectWindow('Channels Stack');
      ChooseSlice(7);
      Paste;
      SurfacePlot;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(14);
      Paste;
      SelectWindow('Average');
      SelectAll;
      GetROI(left,top,width,height);
      MakeLineROI(left,top,left+width-1,top+height-1);
      PlotProfile;
      SelectAll;
      Copy;
      Dispose;
      SelectWindow('Channels Stack');
      SelectSlice(21);
      Paste;
      SelectSlice(7);
      MoveTo(15,15);
      Write('  GREEN & BLUE CHANNELS');
      SelectSlice(14);
      MoveTo(15,15);
      Write('  GREEN & BLUE CHANNELS');
      SelectSlice(21);
      MoveTo(15,15);
      Write('  GREEN & BLUE CHANNELS');
      SelectWindow('Average');
      Dispose;
      Dispose;
      SelectWindow('Example Image');
      Dispose;
      SelectSlice(22);
      MoveTo(30,15);
      SetText('Plain');
      SetFontSize(12);
      WriteLn('  Use the "<" and ">" keys to move Backward and Forward'); 
      WriteLn('  through the Stack.');
      WriteLn('');
      WriteLn('');
      WriteLn('  The Profile Plot runs from the top left corner to the ');
      WriteLn('  bottom right corner.');     
      WriteLn('  You can use the "Plot Profile" tool from the "Tools" Window');
      WriteLn('   (Highlighted) for further manual measurements if you wish. ');
      WriteLn('');
      WriteLn('  Draw across a sample inclusion and some cellular ');
      WriteLn('  background.');
      WriteLn('');
      SaveAs('Channels Stack');
      SelectTool('Profile');
end;
end;

macro '(---'; begin end;

macro '[1] Set Region of Interest';
begin
     SetSaveAs('Outline');
     SaveAs('PASTEHERE');{643}
end;
end;

macro '[2] Calculate Background subtraction';
begin
   GetLine(x1,y1,x2,y2,ignore1);
   if x1<0 then begin
   PutMessage('This macro requires a line selection to determine the rollingball radius.   Use the line selection tool to draw it.');
   exit;
end;
    ResetCounter;
    SetScale(0,'pixel');
  	Measure;
  	Radius := rLength[rCount];
  	PutMessage('RollingBallRadius = ',Radius);
  	PutMessage('Move the rolling ball across image, if at any point the ball is completely (100%) filled by particle then the radius is too small');
  	MakeOvalRoi(x1,y1,Radius,Radius);
end;
end;

macro '(---'; begin end;

macro 'Grey Scale.                                     Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 1000 do begin;
    open('Image.',q:4);
    ApplyROI;
    SubtractBackground('2D Rolling Ball', radius);
    Smooth;
    SelectAll;
    SaveAs('Subtracted.',q:4);
    Dispose;
end;
end;

macro '24 bit TIFF to Grey Scale.           Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 1000 do begin;
    open('Image.',q:4);
    AverageSlices;
    ApplyROI;
    SubtractBackground('2D Rolling Ball',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
    Selectwindow('Image.',q:4);
    Dispose;
end;
end;

macro 'Red Channel.                                  Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    Red;
    ApplyROI;
    SubtractBackground('2D Rolling Ball',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
end;
end;

macro 'Green Channel.                             Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    Green;
    ApplyROI;
    SubtractBackground('2D Rolling Ball',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
end;
end;

macro 'Blue Channel.                                Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    Blue;
    ApplyROI;
    SubtractBackground('2D Rolling Ball',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
end;
end;

macro 'Red and Green Channels.         Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    RedGreen;
    ApplyROI;
    SubtractBackground('2D Rolling Ball',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
    Selectwindow('Image.',q:4);
    Dispose;
end;
end;

macro 'Red and Blue Channels.            Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    RedBlue;
    ApplyROI;
    SubtractBackground('2D Rolling Ball',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
    Selectwindow('Image.',q:4);
    Dispose;
end;
end;

macro 'Green and Blue Channels.       Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    GreenBlue;
    ApplyROI;
    SubtractBackground('2D Rolling Ball',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
    Selectwindow('Image.',q:4);
    Dispose;
end;
end;

macro '(---'; begin end;

macro 'Fast Grey Scale.                                     Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 1000 do begin;
    open('Image.',q:4);
    ApplyROI;
    SubtractBackground('2D Rolling Ball (faster)', radius);
    Smooth;
    SelectAll;
    SaveAs('Subtracted.',q:4);
    Dispose;
end;
end;

macro 'Fast 24 bit TIFF to Grey Scale.           Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 1000 do begin;
    open('Image.',q:4);
    AverageSlices;
    ApplyROI;
    SubtractBackground('2D Rolling Ball (faster)',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
    Selectwindow('Image.',q:4);
    Dispose;
end;
end;

macro 'Fast Red Channel.                                  Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    Red;
    ApplyROI;
    SubtractBackground('2D Rolling Ball (faster)',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
end;
end;

macro 'Fast Green Channel.                             Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    Green;
    ApplyROI;
    SubtractBackground('2D Rolling Ball (faster)',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
end;
end;

macro 'Fast Blue Channel.                                Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    Blue;
    ApplyROI;
    SubtractBackground('2D Rolling Ball (faster)',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
end;
end;

macro 'Fast Red and Green Channels.         Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    RedGreen;
    ApplyROI;
    SubtractBackground('2D Rolling Ball (faster)',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
    Selectwindow('Image.',q:4);
    Dispose;
end;
end;

macro 'Fast Red and Blue Channels.            Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    RedBlue;
    ApplyROI;
    SubtractBackground('2D Rolling Ball (faster)',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
    Selectwindow('Image.',q:4);
    Dispose;
end;
end;

macro 'Fast Green and Blue Channels.       Background Subtraction';
{
Image names must be in the form 'image.0001', 'image.0002', ..., but this can be changed.
}
Var
     q : integer;
begin
    radius:=GetNumber('Rolling ball radius (pixels):',100);
    for q:= 1 to 10000 do begin;
    open('Image.',q:4);
    GreenBlue;
    ApplyROI;
    SubtractBackground('2D Rolling Ball (faster)',radius);
    Smooth;
    SelectAll;
    SetSaveAs('Tiff');
    SaveAs('Subtracted.',q:4);
    Dispose;
    Selectwindow('Image.',q:4);
    Dispose;
end;
end;

macro '(---'; begin end;

Macro '[3] Tell me how to estimate particle size range!'
begin
      PutMessage('First you need to make a stack of some the Background  Subtraction images you have just created. Open four UNINFECTED controls and four INFECTED controls.');
      PutMessage('Once you have opened the appropriate images choose "Windows to Stack" from the Stacks menu. Then save the stack.');
PutMessage('Play about with the Density Slice Settings under the "Options" menu, then choose "[4] Positive and Negative Controls I. Analyze Particles".');
end;
end;

Macro '[4] Positive and Negative Controls I. Analyze Particles'
begin
    CheckForStack;
    NewTextWindow('Controls');
    WriteLn('Particles       Mean Area       Maximum Area          Minimum Area');
    Lower:= GetNumber('Please Enter the Lower Density Slice Value', 80,0);
    Upper:= GetNumber('Please Enter the Upper Density Slice Value', 255,0); 
for i:= 1 to nSlices do begin
    SetParticleSize(1, 10000);
    SelectSlice(i);
    SetScale(0,'pixels');
    ApplyROI;
    SetDensitySlice (Lower, Upper);
    AnalyzeParticles('Outline,Label,Reset');
If (rCount=0) then begin;
     SelectWindow ('Controls');
     WriteLn ('   0                     0                         0                                 0');
End Else Begin;
     Write(rCount);
     MeanAreaValue;
     MaxMin;
end;
end;
      PutMessage('Note the "Maximum Area" values returned for the Uninfected Controls. Set the "minimum Inclusion size" above this, these particles represent normal cellular background.');
      PutMessage('Then look through the stack and see what kind of particles have been counted. Use the < and > keys to move backwards and forwards through the stack.');
     PutMessage('Once you are finished go back to the stack you saved after Positive and negative Controls I. Then run Positive and negative Controls II. Inclusions size.');
end;
end;

Macro '[5] Positive and Negative Controls II . Inclusion Size'
begin
    CheckForStack;
    NewTextWindow('Controls II');
    WriteLn('Inclusions   Mean Area     Maximum Area     Minimum Area                  STDEV');
    Lower:= GetNumber('Please Enter the Lower Density Slice Value', 100,0);
    Upper:= GetNumber('Please Enter the Upper Density Slice Value', 255,0); 
    min:= GetNumber('Please Enter minimum Inclusion size', 25);
    max:= GetNumber('Please Enter maximum Inclusion size', 10000);
for i:= 1 to nSlices do begin
    SetParticleSize(min, max);
    SelectSlice(i);
    SetScale(0,'pixels');
    ApplyROI;
    SetDensitySlice (Lower, Upper);
    AnalyzeParticles('Outline,Label,Reset');
If (rCount=0) then begin;
     SelectWindow ('Controls II');
     WriteLn ('   0                        0                       0                                  0                                    0');
End Else Begin;
     SelectWindow ('Controls II');
     Write(rCount);
      MeanAreaValue;
      MaxMin2;
      Write('             ');
     StandardDeviation2;
end;
end;
      PutMessage('Check through the stack to make sure all the inclusions have been counted. You may need to go back and fine tune the density slice and particle size settings at this point.');
     PutMessage('Once you are happy with the results, and have recorded all the variables you need, you can go on to "Background Subtracted Images.  Automated Inclusion Enumeration".');
end;
end;

Macro '[6] Set Scale part 1'
begin
   PutMessage('Open an image of a graticule or other object of known dimensions at the same magnification as the images you wish process in this session.');
   Open('');
   PutMessage('This macro requires a line selection to set the scale. Use the line selection tool to trace a known distance on the graticule. Then run macro "Set Scale part 2".');
end;
end;

Macro '[7] Set Scale part 2'
begin
PutMessage('Before you proceed make sure that you have run the macro "Set Scale part 2" or completed the steps outlined therein. Fill in the KNOWN DISTANCE and Record the SCALE. '); 
SetScale(0, '' );
end;
end;

macro '(---'; begin end;

Macro '[8] Background Subtracted Images.  Automated Inclusion Enumeration';
{
Image names must be in the form 'Subtracted.0001', 'Subtracted.0002', ..., but this can be changed.
}
begin
    ImageScale:=GetNumber('What is the scale of the images:', 1.125);
    ImageUnits:=GetString('What are the units of measurement:', ' µm2');
    Lower:= GetNumber('Please Enter the Lower Density Slice Value', 100,0);
    Upper:= GetNumber('Please Enter the Upper Density Slice Value', 255,0);
    min:= GetNumber('Please Enter minimum Inclusion size', 50);
    max:= GetNumber('Please Enter maximum Inclusion size', 10000);
    NewTextWindow('Inclusions');
    SaveAs('Inclusions');
    Write('Inclusions       Area', ImageUnits '           STDEV');
    ROISize;
for q:= 1 to 10000 do begin;
    open('Subtracted.',q:4);
    SetScale(ImageScale, '      ImageUnits');
    SetParticleSize(min, max);
    ApplyROI;
    SetDensitySlice (lower, upper);
    MakeBinary;
    ApplyROI;
    DeleteTouching;
    ApplyROI;
    erode;
    dilate;
    AnalyzeParticles('Outline,Label,Reset');
If (rCount=0) then begin;
    SelectWindow ('Inclusions');
    WriteLn ('   0                  0                        0');
End Else Begin;
    SelectWindow ('Inclusions');
    Write(rCount);
    MeanAreaValue;    
    StandardDeviation;
    Save;
end;
     SelectWindow ('Subtracted.',q:4);
     SelectAll;
     SaveAs ('Binary.' ,q:4);
     Dispose;
end;
end;