Michael Hooker Microscopy Facility (MHMF.ORG)

Zeiss 510 - Running Spectral Scans on the Meta

2.  Performing Meta demixing

• Considerations
  • Spectral response will be changed by the following factors in the microscope, therefore must reproduce these factors when doing spectral scanning with different samples and over sessions
    • Laser excitation wavelengths used
    • Immersion oil
    • Objective
    • Dichroic (excitation)
    • Other filters
    • 8 bit/12 bit pixel intensity depth
    • pin hole
  • Fluorophores should not be dim
  • Fluorophore intensity should be not too dissimilar
  • Detector should not be over or under loaded
  • Fluorescent saturation must be avoided, i.e. too much excitation intensity
  • Used laser(s) should be warmed up
  • Can do autofluorescence correction of have sample or region auto-fluorescence alone
• Setup with multiply dyed sample first, then scan single labeled samples

• Lambda mode
• Choose dichroic
• Activate appropriate laser
• 1 <= pinhole <= 2
• Scan speed ideally 6 or 7 and less than equal 10
• Set mim/max on 1:1 view with range indicator on while viewing brightest channel
• Best to scan using xy-scan button - get glow over/under indicator
• Save multiple labeled
• Save single labeleds
• Process -> Unmix -> choose
  • ?     autoscale - no if ratio intensity
  • always yes - advanced
  • yes generate residuals - pixels which can not be assigned to a dye
  • ?    Ask for background - good for reflection control or if some frames should be black but are not

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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