Phase calibration pictures 3/9/10. Dark count image Required for: 1. determining image intensity in...

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