AlanFung:LabNotes/Rolony/2013-9-26
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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)
- 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
Original Image | Intensity Histogram | >5000 Binary |