AlanFung:LabNotes/Rolony/2013-9-26: Difference between revisions

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>Alan6017518
 
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*Call a base based on the signal area
*Call a base based on the signal area
*Generate the 3 base sequence based on the location of the signal
*Generate the 3 base sequence based on the location of the signal
==Tools==
==Tools==
*Images (taken with Hosuk)
*Images (taken with Hosuk)
Line 21: Line 22:


==Protocol==
==Protocol==
*I picked the image as a test (cycle3 Cy3)
*We will use cycle#3 Cy3 image as an example and optimize the image analysis setting
*I=imread('sbs_cycle03_Cy3.tif') ;load the image
*I=imread('sbs_cycle03_Cy3.tif') ;load the image
*DI=double (I); Convert the image to a double class (when storing use single variable as it takes less memory, for processing convert variable to double)
*DI=double (I); Convert the image to a double class (when storing use single variable as it takes less memory, for processing convert variable to double)
*whos ;Check that the variable indeed was converted into double
*whos ;Check that the variable indeed was converted into double
*Hist(DI);
*Hist(DI); Display histogram of intensities of the image
*[[File:https://www.dropbox.com/s/6ihbw1cmoph8ih2/0711_SBS_1st_C03Cy3_Hist.jpg]]
*Looks like the cutoff is at 5000
*Looks like the cutoff is at 5000
*BI = DI > 5000; threshold; choose threshold based on whatever
*iptsetpref('ImshowBorder','tight'); display image without boarder
<!--Start of table definition-->
{|style="background:transparent; color:black" border="0" height="230" align="center" valign="bottom" cellpadding=10px cellspacing=0px
<!--Start of the details for the top-of-table heading-->
|+style="background:transparent; color:black"|'''''Images'''''
<!--Start the first row and add row formats-->
|-align="center"
 
<!--Start of individual cell details for the first row-->
|
|[[Image:sbs_cycle03_Cy3.png|border|300x300px|none]]
|
|[[Image:0711_SBS_1st_C03Cy3_Hist_Cutoff.png|border|300x300px|none]]
|
|[[Image:0711_SBS_1st_C03Cy3_Cutoff.png|border|300x300px|none]]
|
<!--Start of a second row and add formats-->
|-align="center" valign="top"
<!--Start of individual cell details for the second row-->
|width="25"|
|width="100"|'''Original Image'''
|width="25"|
|width="100"|'''Intensity Histogram'''
|width="25"|
|width="100"|'''>5000 Binary'''
|width="25"|
|}

Latest revision as of 00:55, 27 September 2013

Strip off dye coupling with formamide[edit]

  • Aspirate PBS from mattek dish with primary/secondary rolonies
  • Add 200ul preheated (65C-80C) 80% formamide in 2X SSC
  • Incubate 10min RT
  • Rinse w H2O
  • Add in 1:200-1uM 200ul (preheat 65C) ACTB-primary rolony target probe (Alexa 488) (200uM ACTB_1stRolonyFISH)
  • Incubate 10min RT
  • Rinse w 2x SSC twice
  • Add in 2X ssc
  • image

Matlab Image Analysis[edit]

Goal[edit]

  • The goal is to analyze the image taken from SBS sequencing of the primary rolony
  • I would create a threshold to screen out the noise
  • Call a base based on the signal area
  • Generate the 3 base sequence based on the location of the signal

Tools[edit]

  • Images (taken with Hosuk)

[1]

  • Matlab

Protocol[edit]

  • We will use cycle#3 Cy3 image as an example and optimize the image analysis setting
  • I=imread('sbs_cycle03_Cy3.tif') ;load the image
  • DI=double (I); Convert the image to a double class (when storing use single variable as it takes less memory, for processing convert variable to double)
  • whos ;Check that the variable indeed was converted into double
  • Hist(DI); Display histogram of intensities of the image
  • Looks like the cutoff is at 5000
  • BI = DI > 5000; threshold; choose threshold based on whatever
  • iptsetpref('ImshowBorder','tight'); display image without boarder
Images
Original Image Intensity Histogram >5000 Binary