Table 2 Sources of variability in a quantitative optical microscopy pipeline and methods for monitoring and assuring data quality

Sample Preparation

-Establish well-defined protocols for handling cells (ASTM F2998)

Cell culture variability (cell type, donor, passage, history, culturing protocol, user technique)

[23, 94]

-Use stable and validated stains (e.g. photostable, chemically stable, high affinity, well characterized antibody reagents)

Instability of probe molecule and non-specific staining

[95–98]

-Choose substrate with low and homogeneous background signal for selected imaging mode or probe (ASTM F2998)

Interference from background

[94, 99–101]

-Optimize medium [filter solutions to reduce particulates, reduce autofluorescence (phenol red, riboflavin, glutaraldehyde, avoid proteins/serum during imaging)

-Optimize experimental design to the measurement (e.g., low seeding density if images of single cells are best) (ASTM F2998)

Interference from cells in contact

[94, 102]

Image Capture

-Use optical filters to assess limit of detection, saturation and linear dynamic range of image capture (ASTM F2998)

Instrument performance variability (e.g.) light source intensity fluctuations, camera performance, degradation of optical components, changes in focus)

[94, 103, 104]

-Optimize match of dyes, excitation/emission wavelength, optical filters & optical filters

Poor signal and noisy background

[105, 106]

-Minimize refractive index mismatch of objective, medium, coverslips & slides

-Use highest resolution image capture that is practical (e.g., balance throughput with magnification, balance numerical aperture with desired image depth)

-Calibrate pixel area to spatial area with a micrometer

Changes in magnification

[107, 108]

-Collect flat-field image to correct for illumination inhomogeneity (ASTM F2998)

Non-uniformity of intensity across the microscope field of view

[94, 109–112]