Matt:LabNotes/2013-10-30
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PISA7 Analysis on ACTB Targets of Primary Rolonies
- Images taken by Hosuk:LabNotes/2013-10-29
Images
- Hybridized 3 Probes (1st Rolonies, ACTB Target, RAB7A Target) and Imaging ATTO488: S2_20131022_3Dye_DetachTest_2After_ACTB.tif
- Stripped all dyes and Imaging ATTO488: S2_20131022_Add1ProbeOnlyTest_1Strip_ACTB.tif
- 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