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AlanFung:LabNotes/Rolony/2013-9-26
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==Strip off dye coupling with formamide== *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== ==Goal== *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== *Images (taken with Hosuk) [https://www.dropbox.com/sh/nfjx7r1j6d4l8dp/OJ1OJ0YmT-] *Matlab ==Protocol== *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 <!--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"| |}
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