AlanFung:LabNotes/Rolony/2013-9-26: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 (→Goal) |
||
(17 intermediate revisions by the same user not shown) | |||
Line 15: | Line 15: | ||
*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== | ||
* | *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 | ||
*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)
- 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 |