Hosuk:LabNotes/2013-6-11: Difference between revisions

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=====Image processing=====
=====Image processing=====
*'''Imaris'''
*'''Imaris'''
**for rendering, image processing
*'''Huygens'''
*'''Huygens'''
**for deconvolution and image alinging
*Image processing procedure
**Take raw images data to storage server. Raw images from Confocal is 12 bit
**Open raw images with Imaris, and export to Huygens
**Deconvolution with Huygens, end export back to Imaris (become 32 bit)
**Check images with Imaris, and reduce image size
***Maximum projection --> 2D image from 3D z stack images
***Downsampling to 8 bit
***Reduce resolution from 4000 x 4000 to 1000 x 1000
**Link time series
**Create mask for aligning with Imaris and align images with Huygens
**Convert time to z --> Get one image has pixel towers (stacks) of each time series of each individual rolony signal
**Use genome libraries to match each colors of rolonies to known libraries, without concerning further image processing to remove noise, background and resolve which pixel is real or not.





Revision as of 16:01, 11 June 2013

Day 2

Image processing
  • Imaris
    • for rendering, image processing


  • Huygens
    • for deconvolution and image alinging


  • Image processing procedure
    • Take raw images data to storage server. Raw images from Confocal is 12 bit
    • Open raw images with Imaris, and export to Huygens
    • Deconvolution with Huygens, end export back to Imaris (become 32 bit)
    • Check images with Imaris, and reduce image size
      • Maximum projection --> 2D image from 3D z stack images
      • Downsampling to 8 bit
      • Reduce resolution from 4000 x 4000 to 1000 x 1000
    • Link time series
    • Create mask for aligning with Imaris and align images with Huygens
    • Convert time to z --> Get one image has pixel towers (stacks) of each time series of each individual rolony signal
    • Use genome libraries to match each colors of rolonies to known libraries, without concerning further image processing to remove noise, background and resolve which pixel is real or not.


Rolony - Day 2
  • Cell preparation
    • 100% confluence, and could wait one more day, and split
    • When cells are transferred from 6 well plate
    • 500uL of Trypsin, incubate 5min
    • Add 500uL media, and mix well, and
    • 100uL media in 96 well, or 300~350uL media in glass bottom dish
    • Add 60uL of cell in 96 well or 200uL in glass bottom dish
    • Culture one or two more day


  • BS(PEG)9
    • 20uL in 1mL PBS
    • Add 200uL of BS(PEG)9 mix per one glass bottome dish
    • Add 50uL per one well of 96 well plate


  • CircLigase II
    • Use 2uL of enzyme, but make 200uL mix by doubling all reagents
    • 128uL H2O
    • 20uL 10x Buffer
    • 10uL MnCl2
    • 40uL Betaine
    • 2uL CircLigase II


  • RCA
    • 1~2hr. for RT or 2hr. for CircLigase II would be enough, but
    • incubate over 12 hour