An automated protocol for performance benchmarking a widefield fluorescence microscope

Abstract

Widefield fluorescence microscopy is a highly used tool for visually assessing biological samples and for quantifying cell responses. Despite its widespread use in high content analysis and other imaging applications, few published methods exist for evaluating and benchmarking the analytical performance of a microscope. Easy-to-use benchmarking methods would facilitate the use of fluorescence imaging as a quantitative analytical tool in research applications, and would aid the determination of instrumental method validation for commercial product development applications. We describe and evaluate an automated method to characterize a fluorescence imaging system's performance by benchmarking the detection threshold, saturation, and linear dynamic range to a reference material. The benchmarking procedure is demonstrated using two different materials as the reference material, uranyl-ion-doped glass and Schott 475 GG filter glass. Both are suitable candidate reference materials that are homogeneously fluorescent and highly photostable, and the Schott 475 GG filter glass is currently commercially available. In addition to benchmarking the analytical performance, we also demonstrate that the reference materials provide for accurate day to day intensity calibration. Published 2014 Wiley Periodicals Inc. †

A theoretical‐experimental methodology for assessing the sensitivity of biomedical spectral imaging platforms, assays, and analysis methods

“…To better account for detector performance, the theoretical saturation level was decreased to 11.5 bits of dynamic range (a maximum intensity of 2895), resulting in an accurate match to the experimental data ( Figure 3G). Hence, results from the theoretical sensitivity analysis highly correlate with experimental data given the caveat that the full (claimed) dynamic range of a microscope system may not always be achievable (as has been documented elsewhere [105][106][107]). …”

Survey statistics of automated segmentations applied to optical imaging of mammalian cells

“…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 ] …”

Using the NoiSee workflow to measure signal-to-noise ratios of confocal microscopes

“…Many different tools have been successfully used in the past to measure SNR for different types of microscopes and flow cytometers 4,5,14,15,[19][20][21][22] . The method presented here builds on these approaches and additionally offers a ready-to-follow workflow alongside a freely available analysis macro; it can serve as an easy-to-use tool for facilities and high-end confocal microscopy labs to be integrated into their standard routine.…”