phase calibration pictures 3/9/10. dark count image required for: 1. determining image intensity in...
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Phase calibration pictures 3/9/10
Dark count image
Required for: 1. determining image intensity in the absence of light.2. also required for phase artifact correction
Background phase artifacts due to fixed optics
• Required for background subtraction of images collected with identical optics.
Calibration procedure using a blank image
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raw image dark image
[ [] - ]dark imageblank image
These artifacts appear in all image data (upon close inspection) and can be removed
Application of correction procedure
Removal of phase artifacts
After correction
Properties of phase standard• Correct ring alignment
• Mis-aligned rings
• Day to day instrument validation
10x, brightfield image
PDMS stamp on microscope with ~20 µm high features
Benchmark with mis-alignment of phase rings
Reduced contrast between features when alignment is incorrect
Cells with mis-alignment
• Cells with correct and incorrect alignment
Additional information from other imaging modes of the benchmark
Correct alignment (PH1) and illumination Noisy illumination (room lights on)
Contrast of benchmark affected
Brightfield illumination PH2 (not PH1) ring in illumination path