Macro to compute comet moment from single cell gel electrophoresis data. Coment moment is computed as the integrated density in the comet tail multiplied by the distance from the center of the nucleus to the center of mass of the tail.

The macro requires manual intervention. It assumes all tails are going to the left of the image.

Steps:
1) Rotate the comet if necessary with "R".

2) Duplicate a rectangular selection containing the entire nucleus and the comet.

3) Go to the new window and change the color from grey scale to color with "P" to more easily identify the boundaries of the nucleus.

4) Draw a vertical selection with the measurement tool across the width of the nucleus and hit "N" to locate the center and radius of the nucleus. The macro will draw a circle which should cover the nucleus. Repeat if not correct.

5) Make a rectangular selection isolating the comet tail and hit "C". The macro will then subtract the background and compute the comet moment.

6) Please note that this definition of comet moment is not normalized to the total amount of DNA, as we find that with 8 bit grey scale, the dynamic range is not sufficient to capture both the tail and the nucleus, so we normally let the center of the nucleus become saturated. }

var xcenter,ycenter,radius:integer
Macro 'Change to Palette Color [P]';
Begin
SetPalette ('Spectrum');
End;

macro 'Rotate Comet [R]';
var x1,x2,y1,y2,dx,dy,width,degrees,height:integer;
theta,pi:real;
begin
pi:=3.1419265;
GetLine(x1,y1,x2,y2,width);
if x1<0 then begin
PutMessage('Please draw a line from the center of the nucleus along the comet.');
exit;
end;
dx:=x1-x2;
dy:=y1-y2;
if dx=0 then begin
if dy>=0 then theta:=pi/2 else theta:=3/2*pi
end
else
begin
theta:=arctan(dy/dx);
if dx<0 then theta:=theta+pi;
end;
degrees:=-theta*180/pi;
if degrees>180 then degrees:=-degrees-360;
{ PutMessage(dx,' ',dy,' theta ' ,theta,' ',degrees); }
SelectAll;
ScaleAndRotate(1,1,degrees);
end;
end;

macro 'Nucleus Center [N]';
var
x1,x2,y1,y2,top,left,width,height:integer;
begin
GetLine(x1,y1,x2,y2,width);
if x1<0 then begin
PutMessage('Please draw a line across the nucleus width.');
exit;
end;
xcenter:=x1+(x2-x1)/2;
ycenter:=y1+(y2-y1)/2;
radius:=sqrt(sqr(x2-x1)+sqr(y2-y1))/2;
MakeOvalROI(xcenter-radius,ycenter-radius,radius*2,radius*2);
clear;
MakeOvalROI(xcenter-5,ycenter-5,10,10);
fill;
KillRoi;
end;

macro 'Comet Dimensions [C]';
var
left,top,width,height,pixel:integer;
x,y,txval,tyval,tpix,CometXPos,CometYPos,CometLen:integer;
n,mean,mode,min,max:integer;
moment:real;
begin
SetUser1Label('Comet Length');
SetUser2Label('Moment');
SetOptions('Area,Mean,Min/Max,User1,User2');
SetPrecision(2);
GetRoi(left,top,width,height);
if height=0 then begin
PutMessage('Please select the comet tail area');
exit;
end;
width:=xcenter-(radius+left);
MakeRoi(left,top,width,height);
Invert;
SubtractBackgound('2D Rolling Ball (faster)',50);
Measure;
GetResults(n,mean,mode,min,max);
txval:=0;
tyval:=0;
for x:=left to left+width-1 do begin
for y:=top to top+height-1 do begin
pixel:=GetPixel(x,y);
txval:=txval+ pixel*(x);
tyval:=tyval+pixel*(y);
end;
end;
tpix:=n*mean;
CometXPos:=txval/tpix;
CometYPos:=tyval/tpix;
CometLen:=sqrt(sqr(CometXPos-xcenter)+sqr(CometYPos-ycenter));
rUser1[rCount]:=CometLen;
moment:=n*mean*CometLen;
rUser2[rCount]:=moment;
UpdateResults;
Dispose;
end;

Dr. Herbert Geller, Head
Developmental Neurobiology, NHLBI, NIH
Building 10, Room 8C-213, 10 Center Drive, MSC-1650
Bethesda, MD 20892-1650
Email: gellerh@nhlbi.nih.gov

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