Quality control on fluorescence microscopes

what is it and why should we perform it?
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Published on the 10 February 2020

Quality control is a popular topic on the fluorescence microscopy field, and often mistaken with calibration. In fluorescence imaging, and more importantly in life science laboratories, it is a crucial step to ensure the reliability of results. With the complexity of modern imaging instruments, Quality Control is the basis to true quantitative microscopy.

Quality control has been frequently discussed in the field of fluorescence microscopy.

Recently, a session was dedicated to it during ELMI 2019, the UK Microscopy Quality Control RMS Focused Interest Group was created, followed by the BINA Quality Control and Data Management Working Group in North America, and an ISO norm about optical data of fluorescence confocal microscopes was published (Read more about the norm).

1. Quality control: what are we talking about?

 

Quality control consists in determining, by using appropriate tools, if an instrument fulfills the quality specifications or pre-established requirements, to insure the reproducibility of its performances within a certain margin. This implies to know or to define these specifications or requirements, and to monitor them over time.

Quality control does not allow to turn a fluorescence microscope from an imaging instrument into a metrology one. However, it enables the comparison of results between two fluorescence microscopes, or the measure of performance fluctuations of one microscope over time.

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Quality control” is more appropriate than “calibration” for the field of fluorescence microscopy.

The term “quality control” is more appropriate than “calibration” for the field of fluorescence microscopy. Indeed, calibration consists in comparing measurement values delivered by a device being tested with the values of a calibration standard of known accuracy. This implies the use of standards or reference materials, in general from National Metrology Institutes.

2. Why perform quality control?

 

Fluorescence imaging has become ubiquitous in life sciences laboratories, including those focused on pharmaceuticals, diagnosis, and forensics. In life sciences experiments, two main sources of error are possible: errors coming from the sample preparation, and those coming from the imaging system. Controlling regularly the quality of such instruments allows to remove the bias they introduce on life sciences experiments.

Quality control is one of the numerous missions of a Core Facility. As described by the German Bio-Imaging1, “one of the main tasks of an imaging facility is to monitor and maintain the optimal performance of the microscope system hosted by the facility.”

“One of the main tasks of an imaging facility is to monitor and maintain the optimal performance of the microscope system hosted by the facility.”1

Modern fluorescence imaging systems are complex instruments made of many optical, mechanical, and electronical components. The possibilities of misalignment, malfunction or failure of such instruments increase with the number of components. For example, laser-scanning microscopes (confocal, spinning disk, etc.) tend to fluctuate more than widefield microscopes, simply because they have more components (such as piezo stages, galvo mirrors, lasers, and photomultiplier tubes) that are subject to change2.

The different actors involved in the field of fluorescence microscopy can mutually benefit from controlling the quality of such instruments:

  • Manufacturers: ensure instruments’ specifications and prevent disagreements with customers (sample versus instrument).
  • Core facilities: ensure instruments’ level of performances compatible to the users’ expectations, prevent disagreements with users (sample versus instrument), anticipate instrument failure and reduce machine downtime.
  • For users: ensure the acquisition of reliable data, remove the bias introduced by the instrument, know how the system performances evolve over time, and perform quantitative microscopy.
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Quantitative microscopy is very important for the scientific community as it makes it possible to compare studies performed at different times, at different places, and from different instruments3.

There are many aspects to quality-control in a fluorescence microscope. To go further, the German Bio-Imaging articles provides a table of test measurements for maintaining microscope performance.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5071710/

Sources:

(1) E.Ferrando-May et al.., 79, 463-479 (2016).

(2) A. Dixon, T. Heinlein and R. Wolleschensky, “Standardization and quality assurance in fluorescence measurements II,” Chapter 1, Springer-Verlag, Berlin Heidelberg (2008).

(3) J. Pawley, “The 39 Steps: A cautionary tale of quantitative 3-D fluorescence microscopy,” Biotechniques 28, 884-886 (2000).
J. C. Waters, “Accuracy and precision in quantitative fluorescence microscopy”, The Journal of Cell Biology, 185, 1135-1148 (2009).

Header photo by Gerd Altmann on Pixabay
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