Matt:LabNotes/2013-10-30

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Revision as of 22:50, 31 October 2013 by >Mzcai
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PISA7 Analysis on ACTB Targets of Primary Rolonies

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

  • 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

  • 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

  • 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