{This macro set includes macros from "confocal_macros" for the import
of BioRad .pic files. The compensation macros were written by Michael Binks
to use code written by Richard Herd in "NIH-Image_Scatter" available on the 
NIH-Image ftp server.}



var
  i,n,p,o,l,w,h,s,x,y,m,t:integer;
  angle,corrfactor:real;
  name:string;
  
macro 'Import Biorad MRC 600 Z Series [Z]';
{
Imports a Z series(multiple images per file) from a Biorad MRC 600
confocal microscope.  The width, height and number of images are
extracted from the first 3 16-bit word in the 76 byte header and
the file name is extracted from bytes 18-23 of the header. Note that
the Undo and Clipboard buffers must be set to  at least 384K to work with
the typical 768x512 Biorad images.
}
var
  width,height,nImages,offset,hdrsize,i,start,picsize:integer;
begin
  RequiresVersion(1.50);
  width:=512;
  height:=1;
  offset:=0;
  SetImport('8-bit');
  SetCustom(width,height,offset);
  Import(''); {Read header}
  width:=GetPixel(0,0)+GetPixel(1,0)*256;
  height:=GetPixel(2,0)+GetPixel(3,0)*256;
  nImages:=GetPixel(4,0)+GetPixel(5,0)*256;
  Dispose;
  hdrsize:= 76;
  picsize:=width*height;
  if (width<128) or (width>2048) or (height<128) or (height>2048) or
(nImages<1) or (nImages>256) then begin
    PutMessage('This does not seem to be a Biorad MRC 600 Z Series file.');
    exit;
  end;
  start:=1;
  offset:=HdrSize+(start-1)*PicSize;
  SetImport('8-bit; Invert');
  SetCustom(width,height,offset,nimages);
  Import('');
  for i:=1 to nSlices do begin
    SelectSlice(i);
   { Invert;}
    ChangeValues(0,0,1);
    ChangeValues(255,255,254);
  end;
  {Add SetScale  and SliceSpacing  Calls at this point for calibrating mag factor and 
    Z-series spacing. }
end;


macro 'Merge Split BioRAD [S]';
{Makes a macro that color merges a split screen BioRAD image, as a merged color image.
This macro has been altered from that in "confocal_macros" so that the Right
hand image becomes the Red slice (which suits our system).}

var
  p,w,h,rgb,l: integer
 name1,name: string;

begin
  p:= PidNumber;
  SelectPic(p);
  GetPicSize(w,h);
  name1:=WindowTitle;
  l:=length(name1);
  Delete(name1,(l-2),3);
  MakeRoi(0,0,w/2,h);
  Copy;
  SetNewSize(w/2,h); 
  MakeNewStack(name1,'RGB');
{Change this to read the name of the original file "Original Name", then name the new stack as "Original Name - Unsplit&Merge". }
  rgb:=PicNumber;
  SelectPic(rgb);
  AddSlice;
  Paste;
  SetBackground(255);
  AddSlice;
  SelectPic(p);
  MakeRoi(w/2,0,w/2,h);
  Copy;
  SelectPic(rgb);
  SelectSlice(1);
  Paste;
  SetBackGround (255);
  RGBToIndexed('System LUT Dither');
  ChoosePic(p);
  Dispose;
  SelectWindow('Indexed Color');
  Dispose;
end;

macro '(-' begin end; 
 

{The following procedures are used by the Compensation macros}
 
procedure CheckForStack;
begin
  if nSlices=0 then begin
    PutMessage('This window is not a stack');
    exit;
  end;
end;

procedure CheckForRGB;
begin
  if (nSlices>3) or (nSlices<3) then begin
      PutMessage('This window is not an RGB stack');
    exit;
  end;
end;

procedure InvertStack;
begin
  for i:= 1 to nSlices do begin
    ChooseSlice(i);
    Invert;
  end;
  SelectSlice(1);
end;


procedure CompensateStack;
begin
	{Image that is the source of cross-over is selected and modified
	to represent the proportion of the original which is present in the other
	channel}
	
	GetPicSize(w,h);
	SelectSlice(y);
	Duplicate(WindowTitle);
	SetPicName('Correction');
	p:=PicNumber;
	MultiplyByConstant(corrfactor);
	
	{New RGB stack is made with compensated images (the above modified image
	is subtracted from the other channel)}
	
	SetNewSize(w,h);
	MakeNewStack(name,'comp');AddSlice;AddSlice;
	o:=PidNumber;
	
	ChoosePic(n);
	ChooseSlice(x);SelectAll;Copy;
	ChoosePic(o);ChooseSlice(x);Paste;
	ImageMath('sub',o,p,1,0,o);
	ChoosePic(p);Dispose;
	ChoosePic(n);ChooseSlice(y);SelectAll;Copy;
	ChoosePic(o);ChooseSlice(y);Paste;
end;


procedure MarkAxes;
begin
	MoveTo(20,236);
	writeln('(Green)');
	MoveTo(290,392);
	writeln('(Red)');
end;


procedure FirstScatter;
{Makes scatter plot of stack before compensation}
begin
	name:=WindowTitle;n:=PidNumber;
	GetPicSize(w,h);
	l:=length(name);
	Delete(name,(l-2),3);
	
	InvertStack;
	UserCode('TwoSliceScatter',1,2,0);
	SelectAll;Copy;Dispose;
	SetNewSize(456,456);
	MakeNewStack(name,'scatter');Paste;
	{MoveWindow(600,50);}{Suitable for 20" monitor at high resolution}
	s:=PidNumber;
	
	MoveTo(100,50);
	writeln('Normal');
	MarkAxes;
	SetDensitySlice(1,1);
end;

procedure SecondScatter;
{Makes scatter plot of stack after compensation}
begin
	UserCode('TwoSliceScatter',1,2,0);
	SelectAll;Copy;Dispose;
	ChoosePic(s);AddSlice;Paste;
	SetDensitySlice(1,1);
	MoveTo(100,50);
	writeln('Compensation = ',(corrfactor*100):4:2,' %');
	MarkAxes;
end;

procedure MeasureAngle;
{Measures the angle of displacement of the dot plot from the horizontal}
begin
	SetOptions('Angle Area');
	IgnoreParticlesTouchingEdge(true);
	IncludeInteriorHoles(true);
	LabelParticles(false);
	OutlineParticles(true);
	SetParticleSize(1000,99999);
	AnalyzeParticles;Undo;
	if rCount>1 then begin 
	 	for i:=1 to rCount do begin
			if rArea[i]>m then begin
				m:=rArea[i];
				t:=i;
			end;
		end;
 	end;
end;


macro 'Add Slice to Stack [A]';
begin
	CheckForStack;
	SelectSlice(nSlices);
	AddSlice;
end;

macro 'Invert Stack [I]';
begin
	InvertStack;
end;

macro 'RGB to 8-bit colour [R]';
begin
	CheckForStack;
	CheckForRGB;
	name:=WindowTitle;
	SetBackGround (255);
	RGBToIndexed('System LUT Dither');
	SetPicName(name,'.8bCol');
end;


macro '(-' begin end; 
macro 'Compensation Macro Info [F1]';
begin
	NewTextWindow('Compensation Macro Info');
	SetFont('Helvetica');
	SetFontSize(12);
	writeln('These NIH-Image macros are intended for use with 2 colour fluoresence images acquired from a confocal microscope. They make use of User.p code written by Richard Herd in "NIH-Image_Scatter" which he has made available on the NIH-Image ftp server.');
	writeln('');
	writeln('Their purpose is to simplify post-acquisition compensation for "bleed through" of fluorescent emission between red and green detector channels. For quantitative co-localisation studies such a correction is important to obtain objective results.');
	writeln('');
	writeln('A linear relationship between fluorescence intensity and cross-over between detector channels is assumed (and in our experience is correct).');
	writeln('');
	writeln('This is calculated using a scatter plot of pixel values in green and red images in an RGB stack from a single colour fluorescence control. The calculated compensation is then applied on a pixel by pixel basis to images of two colour samples.'); 
	writeln('');
	writeln('Instructions:');
	writeln('');
	writeln('1. Load macros into NIH-Image_Scatter.');
	writeln('2.	Import confocal images and make RGB stack (leave so that background is black).');
	writeln('3.	Select single colour control image and use items [1] or [3] from Special menu (Press [1] or [3] on keyboard)');
	writeln('4.	Select dual colour image and use items [2] or [4] from Special menu (Press [2] or [4] on keyboard)');
	writeln('');
	writeln('"Green -> Red" ([1] and [2]) will compensate for "bleed through" from the green to the red channel and vice versa.');
	writeln('');
	writeln('Please send any comments or criticisms to m.binks@ucl.ac.uk');
end;

macro '(-' begin end; 

macro 'Measure Green  ->  Red Control [1]';

begin
	corrfactor:=0;angle:=0;w:=0;h:=0;
	x:=1;y:=2;t:=1;
	CheckForStack;
	CheckForRGB;
	
	FirstScatter;
	MeasureAngle;
	
	angle:=(90-rAngle[t])/57.295779513;{Calculates displacement from vertical in radians}
	corrfactor:=sin(angle)/cos(angle);{Calculates ratio of 2 sides of triangle (b/a)}
	
	ChoosePic(n);
	CompensateStack;
	SecondScatter;
	ShowMessage('Angle = ',(90-rAngle[1]),' degrees','\','Compensation factor = ',corrfactor);
end;



macro 'Compensate Green -> Red Sample [2]';

begin
	x:=1;y:=2;
	CheckForStack;
	CheckForRGB;
	
	corrfactor:=GetNumber('Correction Factor = ',corrfactor,4);
	FirstScatter;
	
	ChoosePic(n);
	CompensateStack;
	SecondScatter;
end;


macro '(-' begin end; 


macro 'Measure Red  ->  Green Control [3]';

begin
	corrfactor:=0;angle:=0;w:=0;h:=0;
	x:=2;y:=1;t:=1;
	
	CheckForStack;
	CheckForRGB;
	
	FirstScatter;
	MeasureAngle;
	angle:=(rAngle[t])/57.295779513;{Calculates displacement from horizontal in radians}
	corrfactor:=sin(angle)/cos(angle);
	
	ChoosePic(n);
	CompensateStack;
	SecondScatter;
	ShowMessage('Angle = ',rAngle[1],' degrees','\','Compensation factor = ',corrfactor);
end;



macro 'Compensate Red -> Green Sample [4]';
begin
	x:=2;y:=1;
	
	CheckForStack;
	CheckForRGB;
	
	corrfactor:=GetNumber('Correction Factor = ',corrfactor,4);
	FirstScatter;
	
	ChoosePic(n);
	CompensateStack;
	SecondScatter;
end;