Matt:LabNotes/2013-10-30: Difference between revisions

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===PISA7 Analysis on ACTB Targets of Primary Rolonies===
===PISA7 Analysis on ACTB Targets of Primary Rolonies===
*Images taken by [[Hosuk:LabNotes/2013-10-29]]
*Images taken by [[Hosuk:LabNotes/2013-10-29]]
 
*PISA7 Settings: Gaussian std: 4, upper bound: -3e-4
'''Images'''
'''Images'''
#Hybridized 3 Probes (1st Rolonies, ACTB Target, RAB7A Target) and Imaging ATTO488: S2_20131022_3Dye_DetachTest_2After_ACTB.tif
#Hybridized 3 Probes (1st Rolonies, ACTB Target, RAB7A Target) and Imaging ATTO488: S2_20131022_3Dye_DetachTest_2After_ACTB.tif
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| Best Settings? (Area 4-40, Bkg_mult 9)
| Best Settings? (Area 4-40, Bkg_mult 9)
|-
| 24 Counted
||
| 129 Counted
||
| 50 Counted
|-
|-
| [[File:S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-21_bkgmult9_Overlay.png|450px]]
| [[File:S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-21_bkgmult9_Overlay.png|450px]]
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*Strict settings seems to have missed many bright spots but not sure if it's because they are larger than max_area or other reasons
*Lenient settings is able to identify all the bright spots previously left out but also highlights some small spots that could be just noise
*"Best" settings captures most of the small bright spots. Bright spots not identified are obviously too large and about 3 microns in diameter
====Image 2: Stripped Probes====
*Using "Best" settings from Image 1
  img2_raw=imread('Oct292013/S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB.tif','tiff');
  img2_flt=medfilt2(img2_raw,[3 3]);
  [xsize ysize] = size(img2_raw);
  hi2=prctile(double(reshape(img2_flt,1,xsize*ysize)),99.9);
  img2_flt_doub = double(img2_flt);
  img2_double=img2_flt_doub/hi2;
  mask=ones(xsize,ysize);
  [bw_img2 img2segdata]=PISA7(img2_double,mask);
  area upper: 40
  area lower: 4
  axratio lower: .7
  circ upper: 1.5
  circ lower: .8
  perim conn: 8
  bkgmult lower: 9
  [bw2_count dummy2]=size(img2segdata); (bw2_count = 3)
  comp_img2=zeros(2048,2048,3);
  comp_img2(:,:,1)=img2_double;
  comp_img2(:,:,3)=bw_img2;
  imwrite(comp_img2,'Oct292013/S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB_PISA7_area4-40_bkgmult9_Overlay.png','png');
*Counted 3 rolonies
[[File:S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB_PISA7_area4-40_bkgmult9_Overlay.png|450px]]
====Image 3: Hybridized ACTB probe only====
*Using "Best" settings from Image 1
  img3_raw=imread('Oct292013/S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB.tif','tiff');
  img3_flt=medfilt2(img3_raw,[3 3]);
  [xsize ysize] = size(img3_raw);
  hi3=prctile(double(reshape(img3_flt,1,xsize*ysize)),99.9);
  img3_flt_doub = double(img3_flt);
  img3_double=img3_flt_doub/hi3;
  mask=ones(xsize,ysize);
  [bw_img3 img3segdata]=PISA7(img3_double,mask);
  area upper: 40
  area lower: 4
  axratio lower: .7
  circ upper: 1.5
  circ lower: .8
  perim conn: 8
  bkgmult lower: 9
  [bw3_count dummy3]=size(img3segdata); (bw3_count = 205)
  comp_img3=zeros(2048,2048,3);
  comp_img3(:,:,1)=img3_double;
  comp_img3(:,:,3)=bw_img3;
  imwrite(comp_img3,'Oct292013/S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB_PISA7_area4-40_bkgmult9_Overlay.png','png');
*Less stringent (bkgmult_lower: 7)
  [bw_img4 img4segdata]=PISA7(img3_double,mask);
  area upper: 40
  area lower: 4
  axratio lower: .7
  circ upper: 1.5
  circ lower: .8
  perim conn: 8
  bkgmult lower: 7
  [bw4_count dummy4]=size(img4segdata); (bw4_count = 293)
  comp_img4=zeros(2048,2048,3);
  comp_img4(:,:,1)=img3_double;
  comp_img4(:,:,3)=bw_img4;
  imwrite(comp_img4,'Oct292013/S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB_PISA7_area4-40_bkgmult7_Overlay.png','png');
{| {{table}}
| Best Settings (Area 4-40, Bkg_mult 9)
||
| Less stringent Settings? (Area 4-40, Bkg_mult 7)
|-
| 205 Counted
||
| 293 Counted
|-
| [[File:S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB_PISA7_area4-40_bkgmult9_Overlay.png|450px]]
||
| [[File:S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB_PISA7_area4-40_bkgmult7_Overlay.png|450px]]
|} 
*Looking at the ACTBOnly image, the spots near the right edge 1/3 of the way from the bottom seem to be true ACTB targets and not noise as thought when looking at Image 1
**Maybe best settings are area 4-40 and bkgmult 7

Latest revision as of 01:27, 5 November 2013

PISA7 Analysis on ACTB Targets of Primary Rolonies[edit]

Images

  1. Hybridized 3 Probes (1st Rolonies, ACTB Target, RAB7A Target) and Imaging ATTO488: S2_20131022_3Dye_DetachTest_2After_ACTB.tif
  2. Stripped all dyes and Imaging ATTO488: S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB.tif
  3. Hybridized ACTB Probe and Imaging ATTO488: S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB.tif

Image 1: Hybridized 3 Probes[edit]

  • Strict settings: Area 4-21 pixels, Bkgmult_lower 9
 img1_raw=imread('Oct292013/S2_20131022_3Dye_DetachTest_2After_ACTB.tif','tiff');
 img1_flt=medfilt2(img1_raw,[3 3]);
 [xsize ysize] = size(img1_raw);
 hi1=prctile(double(reshape(img1_flt,1,xsize*ysize)),99.9);
 img1_flt_doub = double(img1_flt);
 img1_double=img1_flt_doub/hi1;
 mask=ones(xsize,ysize);
 [bw_img1 img1segdata]=PISA7(img1_double,mask);
 area upper: 21
 area lower: 4
 axratio lower: .7
 circ upper: 1.5
 circ lower: .8
 perim conn: 8
 bkgmult lower: 9
 [bw1_count dummy1]=size(img1segdata); (bw1_count = 24)
 comp_img1=zeros(2048,2048,3);
 comp_img1(:,:,1)=img1_double;
 comp_img1(:,:,3)=bw_img1;
 imwrite(comp_img1,'Oct292013/S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-21_bkgmult9_Overlay.png','png');
  • Lenient settings: Area 4-199 pixels, Bkgmult_lower 7
 [bw_img1 img1segdata]=PISA7(img1_double,mask);
 area upper: 199
 area lower: 4
 axratio lower: .7
 circ upper: 1.5
 circ lower: .8
 perim conn: 8
 bkgmult lower: 7
 [bw1_count dummy1]=size(img1segdata); (bw1_count = 129)
 comp_img1=zeros(2048,2048,3);
 comp_img1(:,:,1)=img1_double;
 comp_img1(:,:,3)=bw_img1;
 imwrite(comp_img1,'Oct292013/S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-199_bkgmult7_Overlay.png','png');
  • Best settings?: Area 4-40 pixels, Bkgmult_lower 9
 [bw_img1 img1segdata]=PISA7(img1_double,mask);
 area upper: 40
 area lower: 4
 axratio lower: .7
 circ upper: 1.5
 circ lower: .8
 perim conn: 8
 bkgmult lower: 9
 [bw1_count dummy1]=size(img1segdata); (bw1_count = 50)
 comp_img1=zeros(2048,2048,3);
 comp_img1(:,:,1)=img1_double;
 comp_img1(:,:,3)=bw_img1;
 imwrite(comp_img1,'Oct292013/S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-40_bkgmult9_Overlay.png','png');
Strict Settings (Area 4-21, Bkg_mult 9) Lenient Settings (Area 4-199, Bkg_mult 7) Best Settings? (Area 4-40, Bkg_mult 9)
24 Counted 129 Counted 50 Counted
File:S2 20131022 3Dye DetachTest 2After ACTB PISA7 area4-21 bkgmult9 Overlay.png File:S2 20131022 3Dye DetachTest 2After ACTB PISA7 area4-199 bkgmult7 Overlay.png File:S2 20131022 3Dye DetachTest 2After ACTB PISA7 area4-40 bkgmult9 Overlay.png
  • Strict settings seems to have missed many bright spots but not sure if it's because they are larger than max_area or other reasons
  • Lenient settings is able to identify all the bright spots previously left out but also highlights some small spots that could be just noise
  • "Best" settings captures most of the small bright spots. Bright spots not identified are obviously too large and about 3 microns in diameter

Image 2: Stripped Probes[edit]

  • Using "Best" settings from Image 1
 img2_raw=imread('Oct292013/S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB.tif','tiff');
 img2_flt=medfilt2(img2_raw,[3 3]);
 [xsize ysize] = size(img2_raw);
 hi2=prctile(double(reshape(img2_flt,1,xsize*ysize)),99.9);
 img2_flt_doub = double(img2_flt);
 img2_double=img2_flt_doub/hi2;
 mask=ones(xsize,ysize);
 [bw_img2 img2segdata]=PISA7(img2_double,mask);
 area upper: 40
 area lower: 4
 axratio lower: .7
 circ upper: 1.5
 circ lower: .8
 perim conn: 8
 bkgmult lower: 9
 [bw2_count dummy2]=size(img2segdata); (bw2_count = 3)
 comp_img2=zeros(2048,2048,3);
 comp_img2(:,:,1)=img2_double;
 comp_img2(:,:,3)=bw_img2;
 imwrite(comp_img2,'Oct292013/S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB_PISA7_area4-40_bkgmult9_Overlay.png','png');
  • Counted 3 rolonies

File:S2 20131022 Add1ProbeOnlyTest 1Strip ACTB PISA7 area4-40 bkgmult9 Overlay.png

Image 3: Hybridized ACTB probe only[edit]

  • Using "Best" settings from Image 1
 img3_raw=imread('Oct292013/S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB.tif','tiff');
 img3_flt=medfilt2(img3_raw,[3 3]);
 [xsize ysize] = size(img3_raw);
 hi3=prctile(double(reshape(img3_flt,1,xsize*ysize)),99.9);
 img3_flt_doub = double(img3_flt);
 img3_double=img3_flt_doub/hi3;
 mask=ones(xsize,ysize);
 [bw_img3 img3segdata]=PISA7(img3_double,mask);
 area upper: 40
 area lower: 4
 axratio lower: .7
 circ upper: 1.5
 circ lower: .8
 perim conn: 8
 bkgmult lower: 9
 [bw3_count dummy3]=size(img3segdata); (bw3_count = 205)
 comp_img3=zeros(2048,2048,3);
 comp_img3(:,:,1)=img3_double;
 comp_img3(:,:,3)=bw_img3;
 imwrite(comp_img3,'Oct292013/S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB_PISA7_area4-40_bkgmult9_Overlay.png','png');
  • Less stringent (bkgmult_lower: 7)
 [bw_img4 img4segdata]=PISA7(img3_double,mask);
 area upper: 40
 area lower: 4
 axratio lower: .7
 circ upper: 1.5
 circ lower: .8
 perim conn: 8
 bkgmult lower: 7
 [bw4_count dummy4]=size(img4segdata); (bw4_count = 293)
 comp_img4=zeros(2048,2048,3);
 comp_img4(:,:,1)=img3_double;
 comp_img4(:,:,3)=bw_img4;
 imwrite(comp_img4,'Oct292013/S2_20131022_Add1ProbeOnlyTest_2AddACTBOnly_ACTB_PISA7_area4-40_bkgmult7_Overlay.png','png');
Best Settings (Area 4-40, Bkg_mult 9) Less stringent Settings? (Area 4-40, Bkg_mult 7)
205 Counted 293 Counted
File:S2 20131022 Add1ProbeOnlyTest 2AddACTBOnly ACTB PISA7 area4-40 bkgmult9 Overlay.png File:S2 20131022 Add1ProbeOnlyTest 2AddACTBOnly ACTB PISA7 area4-40 bkgmult7 Overlay.png
  • Looking at the ACTBOnly image, the spots near the right edge 1/3 of the way from the bottom seem to be true ACTB targets and not noise as thought when looking at Image 1
    • Maybe best settings are area 4-40 and bkgmult 7