ImagePro Premier macro for intensity measurement of multiple ROIs

inexperienced

Hello,

I am not good at macro scripting in ImagePro Premier. I used such a macro (see below) with ImagePro Plus 7 to obtain a time sequence of total intensity of multiple ROIs, each of which contained a fluorescent spot, created by the manual tag tool.

The macro mainly consists of the following four parts.

(1) Multiple points were selected in the upper half of a sequence (movie).

(2) The xy coordinates of the counterpart points in the lower half of a sequence were calculated (+ 63 pixel (y coordinate)).

(3) Square ROIs (9x9 pixels) were created using the points.

(4) Intensity of the ROIs were calculated and saved in a file.

Could you give me some advice or hopefully write a macro that works for ImagePro Premier?

Thank you in advance.

Option Explicit
Sub fi_counterpart()
 Dim i As Integer
 Dim frame0 As Long
 Dim frame1 As Long
 Dim FrLen As Integer
 Dim FrStart As Integer
 Dim Radius As Integer
 Dim filename As String * 255
 ret=IpTagShow(1)
'
 Dim dlg As UserDialog
 Dialog dlg
'
 Begin Dialog UserDialog 450, 100, "Title Line"
  Text 10, 10, 400, 20, "Do you want to add Title line?"
  PushButton 150,60,50,20, "Yes"
  PushButton 250,60,50,20, "No"
 End Dialog
 Dim dlg2 As UserDialog
'
 ret = IpSeqGet( SEQ_ACTIVEFRAME, frame0)
 ret = IpSeqGet( SEQ_END,         frame1)
'
 ret = IpStGetInt("Length of Run (Frame)", FrLen, CInt( (frame1 - frame0 + 1) ), 1, CInt( (frame1 - frame0 + 1) ))
 If ret = 0 Then End
 ret = IpStGetInt("Start Time (Frame)", FrStart, 0, 0, 10000)
 If ret = 0 Then End
 ret = IpStGetInt("ROI Size to Measure (Pixel)", Radius, 4, 1, 15)
 If ret = 0 Then End

 Dim SHIFT_X As Integer
 Dim SHIFT_Y As Integer
 ret = IpStGetInt("SHIFT x (pixel)", SHIFT_X, 0, -640, 640)
 If ret = 0 Then End
 ret = IpStGetInt("Shift y (pixel)", SHIFT_Y, 63, -480, 480)
 If ret = 0 Then End
 ret = IpStGetName("Specify filename for output", CurDir$(), "*.dat", filename)
 If ret = 0 Then End
'
 Dim PtsIndex As Integer
 Dim TotalPts As Integer
 Dim nPts As Single
 Dim lpParam(7) As Single
 Dim PtCrd() As RECT
'
 Dim PtCrd_counterpart() As RECT

'
 ret = IpTagGet( GETNUMPTS, -1, nPts)
 TotalPts = nPts
' Total number of tags = nPts
 ReDim PtCrd( TotalPts ) As RECT
 ReDim PtCrd_counterpart( TotalPts ) As RECT
 For PtsIndex = 0 To (TotalPts - 1)
  ret = IpTagGet(GETPOINTS, PtsIndex, lpParam(0))
  PtCrd( PtsIndex ).Left  = CInt(lpParam(0)) - Radius
  PtCrd( PtsIndex ).Right = CInt(lpParam(0)) + Radius
  PtCrd( PtsIndex ).top   = CInt(lpParam(1)) - Radius
  PtCrd( PtsIndex ).bottom= CInt(lpParam(1)) + Radius

  PtCrd_counterpart( PtsIndex ).Left  = CInt(lpParam(0)) - Radius  + SHIFT_X
  PtCrd_counterpart( PtsIndex ).Right = CInt(lpParam(0)) + Radius  + SHIFT_X
  PtCrd_counterpart( PtsIndex ).top   = CInt(lpParam(1)) - Radius  + SHIFT_Y
  PtCrd_counterpart( PtsIndex ).bottom= CInt(lpParam(1)) + Radius  + SHIFT_Y
  Debug.Print PtsIndex
 Next PtsIndex
'
 Open filename For Append As #1
 If ( Dialog(dlg2) = 1 ) Then
  Print #1, "Frame Time";
  For PtsIndex = 0 To (TotalPts-1)
   Print #1, " " ; Format$(PtsIndex, "R000") ;
  Next PtsIndex
  Print #1, ""
 End If
'
 For i = 1 To FrLen
  Print #1, (FrStart + i - 1) ; " " ; Format( (CSng(FrStart + i - 1) / 30.0), "####0.000") ;
'  ret = IpTagUpdate()
  Call FiRead_counterpart(TotalPts, PtCrd(), PtCrd_counterpart())
  ret = IpSeqPlay(SEQ_NEXT)
 Next i
 Close #1
 End
End Sub

Sub FiRead_counterpart(TotalPts As Integer, PtCrd() As RECT, PtCrd_counterpart() As RECT)
 Dim i As Integer
 Dim j As Integer
 Dim k As Integer
 Dim l As Integer
 Dim m As Integer
 Dim imgbuff() As Integer
 Dim fi As Long
 Dim fi_counterpart As Long
 Dim dx As Integer
 Dim dy As Integer
 Dim dx_counterpart As Integer
 Dim dy_counterpart As Integer
'
 dx = PtCrd(0).Right - PtCrd(0).Left + 1
 dy = PtCrd(0).bottom - PtCrd(0).top + 1
 dx_counterpart = PtCrd_counterpart(0).Right - PtCrd_counterpart(0).Left + 1
 dy_counterpart = PtCrd_counterpart(0).bottom - PtCrd_counterpart(0).top + 1
 ReDim imgbuff(dx, dy)
'
 For i = 0 To (TotalPts-1)
  IpDocGetArea( DOCSEL_ACTIVE, PtCrd( i ), imgbuff(0, 0), 0)
  fi = 0
  For j = 0 To dx
   For k = 0 To dy
    fi = fi + imgbuff(j, k)
   Next k
  Next j

  IpDocGetArea( DOCSEL_ACTIVE, PtCrd_counterpart( i ), imgbuff(0, 0), 0)
  fi_counterpart = 0
  For l = 0 To dx_counterpart
   For m = 0 To dy_counterpart
    fi_counterpart = fi_counterpart + imgbuff(l, m)
   Next m
  Next l
  Print #1, " " ; Format(fi, "######");
  Print #1, " " ; Format(fi_counterpart, "######");
 Next i
 Print #1, Chr$(13);
End Sub

YuriG

inexperienced,

Getting intensity measurements from multiple ROIs in Image-Pro Premier is much easier than in IPPlus. We have a special tool "Intensity Tracking" (Measure tab) specifically for that (you don't need a macro). You just draw measurement areas on the image and the intensity distributions are reported in data tables and graphs:



Yuri

inexperienced

Yuri,

Thank you for your response and sorry for the delay in writing this comment.

I know the “Intensity Tracking” tool, however, this tool seems not to draw counterpart ROIs automatically.

What I would like to learn is the way how a macro obtains xy coordinates of manual tags and ROIs (the coordinates are displayed in the Data Table and Feature collections etc.) and create new ROIs using these coordinates.

Intensity measurement of multiple ROIs, which I would like to do, may be performed by manual tags and ROIs.

(1) By manual tags

(1-1) Select multiple points (fluorescent spots) by manual tagging containing the fluorescent spots in the upper half of a sequence (movie).

(1-2) The xy coordinates the manual tags are obtained and the counterpart fluorescent spots in the lower half of a sequence are calculated (for example, + 63 pixel (y coordinate)) using the coordinates.

(1-3) Square ROIs are created using the obtained and calculated coordinates as centers.

(1-4) Intensity of the ROIs are measured by the Intensity Tracking tool and saved in a file.

(2) By ROIs

(2-1) Load an roi file containing multiple square ROI collection.

(2-2) Move the ROIs to fluorescent spots in the upper half of a sequence (movie).

(2-3) The xy coordinates of the counterpart fluorescent spots in the lower half of a sequence are calculated (for example,+ 63 pixel (y coordinate)) using the coordinates of the ROIs.

(2-4) Square ROIs for the counterpart fluorescent spots are created using the coordinates.

(2-5) Intensity of the moved and created ROIs are measured by the Intensity Tracking tool and saved in a file.

inexperienced

 

YuriG

inexperienced,

I have missed your responce and found it only today. Here is what I can recommend:

1. You can get coordinates of measurement objects/tags using GetFeaturePoints function, please check this forum post as an example: http://forums.mediacy.com/discussion/comment/2311#Comment_2311 
2. Creation of objects: (ROI, Measurements, Tracking areas) are macro-recordable, so you can just activate macro recording, create a test objects and stop recording, then you can modify the recorded macro to use the coordinates obtained by the step 1.

Yuri

inexperienced

Yuri

 Thank you for your reply.

 I can get coordinates of measurement tags.

 I still have to do to complete my task.

1) Create counterpart tags

2) ROIs or tracking areas are created using the coordinates of tags

3) Intensity of the ROIs or tracking areas are measured

I am glad if you would give some suggestions for them.

inexperienced

YuriG

inexperienced,

All your tasks, from 1 to 3, can be coded based on auto-recorded macros. You just activate macro recording and do these steps, then you can modify the macro to use coordinates of measurement tags. 

Yuri