International Test Results for Objective Lens Quality, Resolution, Spectral Accuracy and Spectral Separation for Confocal Laser Scanning Microscopes

Abstract

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

Advanced light microscopy core facilities: Balancing service, science and career

“…Regular system checks should be common practice and measure e.g. the point spread function (PSF), the laser power, the illumination homogeneity of the field of view, the detector sensitivity as signal‐to‐noise‐ratio (SNR) and the coefficient of variation (Cole et al, 2011, 2013; Gelman and Rietdorf, 2010; Hibbs, 2006; Stack et al, 2011; Theer et al, 2014; Zucker and Price, 2001, 1999). When performed regularly, standardized test series often help identify damaged and misaligned microscope components, even before they significantly impair the quality of user data.…”

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

“…This process includes quality control on individual systems over time as well as inter-system comparisons. Many criteria can/should be routinely tested, such as illumination uniformity across FOV (using a fluorescein solution, or following the protocol described by Brown and coworkers 16 ), XYZ chromatic aberrations and resolution (using the PSFj 17 or MetroloJ 18 tools, or using 0.2 µm beads as detailed by Cole and colleagues 19,20 ), laser power and stability (using power meter/time series). Maintaining a well-adjusted system by monitoring the SNR is an important step that can give valuable information about the quality of the system, its proper alignment, detector sensitivity and overall system status, especially as the SNR is the limiting factor in image quality and ultimately limits resolution in microscopy.…”

“…This process includes quality control on individual systems over time as well as inter-system comparisons. Many criteria can/should be routinely tested, such as illumination uniformity across FOV (using a fluorescein solution, or following the protocol described by Brown and coworkers 16 ), XYZ chromatic aberrations and resolution (using 0.2 µm beads for instance, nicely detailed by Cole and colleagues 17,18 ), laser power and stability (using power meter/time series).…”

Modern Laser Scanning Confocal Microscopy

“…One downside to spectral array PMTs however is their increased cost, and that they require meticulous calibration for optimal performance (Cole et al, 2013). Spectral imaging uses the detection array to record the emission of a fluorophore throughout the entire visible region of the electromagnetic spectrum, thus generating what is termed a 'λ-stack.'…”