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>Mzcai (Created page with "===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 Ta...") |
>Mzcai |
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(6 intermediate revisions by the same user not shown) | |||
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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 | ||
Line 63: | Line 63: | ||
comp_img1(:,:,3)=bw_img1; | comp_img1(:,:,3)=bw_img1; | ||
imwrite(comp_img1,'Oct292013/S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-40_bkgmult9_Overlay.png','png'); | imwrite(comp_img1,'Oct292013/S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-40_bkgmult9_Overlay.png','png'); | ||
{| {{table}} | |||
| 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|450px]] | |||
|| | |||
| [[File:S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-199_bkgmult7_Overlay.png|450px]] | |||
|| | |||
| [[File:S2_20131022_3Dye_DetachTest_2After_ACTB_PISA7_area4-40_bkgmult9_Overlay.png|450px]] | |||
| | |||
|} | |||
*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 taken by Hosuk:LabNotes/2013-10-29
- PISA7 Settings: Gaussian std: 4, upper bound: -3e-4
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[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