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====Initial Imaging==== *Using PISA7 Paramters: Gaussian std (3), upper bound (-2e-4), area upper (50), area lower (4), axratio lower (.6), circ upper (1.6), circ lower (.8), perim conn (8), bkgmult lower (variable) *First used PISA to examine the images with varying bkgmult lower (range: 2-9) {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Bkgmult''' | align="center" style="background:#f0f0f0;"|'''Rolony Count''' | align="center" style="background:#f0f0f0;"|'''Notes''' |- | NoDye_G680_Cy3||7||116|| |- | NoDye_G680_Cy3||9||31|| |- | NoDye_L2%_G680_FAM||7||202||picked up mostly nuclei |- | NoDye_L2%_G680_FAM||9||7|| |- | dcProbe0-Cy3_G550||7||178|| |- | dcProbe0-Cy3_G550||9||162|| |- | dcProbe0-Cy3_G550||5||208|| |- | dcProbe0-Cy3_G550||3||265||decreasing bkgmult detects more "true" features without significantly counting artifacts |- | dcProbe0-Cy3_G550||2||278||doesn't detect anymore "true" features, just adds noise |- | dcProbe0-Cy3_G680||7||544|| |- | dcProbe0-Cy3_G680||9||489|| |- | dcProbe0-FAM_L5%_G550||7||129|| |- | dcProbe0-FAM_L5%_G550||3||209|| |- | dcProbe0-FAM_L5%_G550||2||245||still misses many while counting artifacts |- | dcProbe0-FAM_G550||7||52|| |- | dcProbe0-FAM_G550||5||68|| |- | dcProbe0-FAM_G550||3||108||even lowering it to 3 still won't count obvious features |- | dcProbe0-FAM_G600||7||313|| |- | dcProbe0-FAM_G600||5||364||good |- | dcProbe0-FAM_G680||7||519|| |- | dcProbe0-FAM_G680||9||375||not bad, pretty similar to G600 bkgmult5 |} '''Count Cells in Field of View''' *NoDye_L2%_G680_FAM had nuclei with high intensity fluorescence relative to background, which was really obvious after processing in PISA so counted nuclei in that image **110 Nuclei/Cells counted [[File:Agi26k0gap_PGP1f_InitialDetect_NucleiCount110.png|450px]] '''Best Images and PISA Settings''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Image Name''' | align="center" style="background:#f0f0f0;"|'''Image''' | align="center" style="background:#f0f0f0;"|'''Feature Count''' |- | Cy3 Background||NoDye_G680_Cy3_Bkgmult9||[[File:NoDye_G680_Cy3_ch00_PISA7Overlay_Bkgmult9.png|450px]]||31 |- | FAM Background||NoDye_L2%_G680_FAM_Bkgmult9||[[File:NoDye_L2%25_G680_FAM_ch00_PISA7Overlay_Bkgmult9.png|450px]]||7 |- | dcProbe0-Cy3||dcProbe0Cy3_G550_Bkgmult3||[[File:DcProbe0Cy3_G550_Cy3_ch00_PISA7Overlay_Bkgmult3.png|450px]]||265 |- | dcProbe0-FAM||dcProbe0FAM_G600_Bkgmult5||[[File:DcProbe0FAM_600_FAM_ch00_PISA7Overlay_Bkgmult5.png|450px]]||364 |} '''Removing Background''' *'''dcProbe0-Cy3''' Ran PISA on NoDye_G680_Cy3_Bkgmult9 and dcProbe0Cy3_G550_Bkgmult3 Saved bw images in workspace as backgroundCy3 and dcProbe0Cy3 countOverlappedPoloniesv2(backgroundCy3,dcProbe0Cy3) ans = 0 comp_img1=zeros(xsize,ysize,3); comp_img1(:,:,1)=dcProbe0Cy3; comp_img1(:,:,2)=backgroundCy3; imwrite(comp_img1,'dcProbe0Cy3vsbackgroundCy3.png'); [[File:DcProbe0Cy3vsbackgroundCy3.png|450px]] *'''dcProbe0-FAM''' Ran PISA on NoDye_L2%_G680_FAM_Bkgmult9 and dcProbe0FAM_G600_Bkgmult5 Saved bw images in workspace as backgroundFAM and dcProbe0FAM countOverlappedPoloniesv2(backgroundFAM,dcProbe0FAM) ans = 0 comp_img1=zeros(xsize,ysize,3); comp_img1(:,:,1)=dcProbe0Cy3; comp_img1(:,:,2)=backgroundCy3; imwrite(comp_img1,'dcProbe0Cy3vsbackgroundCy3.png'); [[File:DcProbe0FAMvsbackgroundFAM.png|450px]] *Conclusion: background is not a factor '''Specificity of dcProbe0-Cy3 and dcProbe0-FAM''' *Is there overlap between the two? (There shouldn't be, they should be exclusive) countOverlappedPoloniesv2(dcProbe0Cy3,dcProbe0FAM) ans = 1 [[File:DcProbe0Cy3vsdcProbe0FAM.png|450px]] *The only overlap is in the bottom right corner and looks more likely due to nearby rolonies than non-specific binding of both dyes to some structure **Red is dcProbe0-Cy3 and Green is dcProbe0-FAM '''Overlay BrightField'''<br> [[File:DcProbe0Cy3vsdcProbe0FAM_BF.png|650px]]
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