Lipid microdomains

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UNDER CONSTRUCTION

Szeged

Contents

Sample preparation

  • Cultured cells are plated on glass bottom dish and incubated with a fluorescent probe for 15 min on room temperature. After 2x washing in PBS large scans (1mm x 1 mm) or movies are taken with CytoScout fluorescent microscope using the apropiate line of the Ar-Kr laser for exitation.

Instrumentation and method

Ultrasensitive Epifluorescent Microscope

  • Type: CytoScout
  • Exitation: Spectra-Physics 2018-RM Ar-Kr mixed gas laser
  • Cameras: 2 CoolSnap HQ Monochrome camera. 1392 x 1040 imaging array, 6.45 x 6.45 um pixels
  • Microscope: Zeiss Axiovert 200
  • Objectives: Zeiss 20x, 40x, 100x NeoFluar objectives
  • Scanning Stage: Marzhauser (temperature controlled CO2 incubation possible)
  • Auto focus System


Mode of data acquisition

  • High Speed Scanning mode: up to 2 mm x 2 mm scan size with 6.45 nm x 6.45 nm pixel size
  • Movie mode with high time resolution:

Max frame rate: 66 fps Max image size: 1392 x 1040 pixel


Data analysis

  • Acquired scans are filtered by FFT bandpass filter of WCIF ImageJ softvare for eliminating low frequency noise. For detecting, characterizing and tracking of labeled membrane domains open source CellProfiler and Matlab based custom made software routines are used. The domains are sorted into classes according to diameter. Diameters are calculated as 2x(Npix/Pi) 2, where X is the size of a pixel in nanometers, and Npix is the number of pixels covering the actual domain.
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