Commentary and Perspective
Standardization of luminescence, fluorescence measurements, and light microscopy: Current situation and perspectives
2022 年 19 巻 論文ID: e190037
詳細
2022 年 19 巻 論文ID: e190037
Improving reproducibility and confidence are important challenges in biomedical measurements, such as luminescence and fluorescence measurements [1]. In general, biomedical measurement results using luminescence and fluorescence are described in terms of optical signals. Although the quantitative aspects of luminescence, fluorescence measurement, and light microscopy are increasingly critical for experimental outputs, the measurement values are still described in arbitrary units. This makes it difficult to compare the results obtained using different equipment because the factors used to determine the optical signal value depend on the type of measurement system and detector used, the spectral properties of the optical components in the light path, and the day the measurement is taken. Standardizing system calibration and verification procedures and establishing reference materials as a common “scale” support a universal comparison between measurement results obtained under different instruments and conditions, thereby ensuring improved result reproducibility and reliability. At the 60th Annual Meeting of the Biophysical Society of Japan, held in September 2022, we organize a symposium session to introduce this research goal.
Bioluminescence (BL) is biological light emitted from the chemical oxidation of luciferin catalyzed by luciferase. BL optical signal is useful as a readout for life science analytical methods. Thus far, ten luciferins and several corresponding luciferases have been identified in bioluminescent organisms [2]. Among the ten choices, BL systems based on firefly luciferin, cypridinid luciferin, and coelenterazine have been commercialized and applied in reporter assays, in vivo and in vitro imaging, and immunoassays [3]. For example, the firefly luciferin-luciferase reaction has been mainly used in gene expression analysis as a reporter assay for screening bioactive compounds or toxicants in vitro [4]. This reaction has also been applied to evaluate cellular functions in vitro, cancer growth, and in vivo tracking. However, it is not easy to directly compare the optical signals of different bioluminescent systems because the colors range from blue to red and are relative values. Instead, it is necessary to standardize the optical signals of different systems as an absolute value based on the same light “scale”. To quantify the light signals from different equipment in BL imaging (BLI), the imaging equipment was first calibrated using a reference LED light source with pulse-width modulation [4]. It is important to evaluate the total radiant flux (W) of this light source linked to a national light standard. The BLI can be normalized from the relative light signal (RLU/pixel) to the absolute light signal (photons/pixel and photons/μm2) [5]. For instance, green- and red-emitting beetle luciferases expressed in mammalian cells controlled by a constitutive promoter produce the difference color light signal. Based on the absolute sensitivity and light collection efficiency, the total absolute optical signal ranged from several thousand photons/second at the single-cell level are determined. This is an extremely weak light intensity corresponding to the attowatt level. Using a reference light source, we can directly compare BLI taken on different days or with different probes and equipment.
Fluorescence microscopy is employed globally as a general method to study biomolecule localization and distribution in cells and tissues in both life sciences and medical research fields. This system is being applied for quantitative measurements in systems biology, high-content image analysis, and imaging-based drug screening. In such fields, fluorescence microscope systems are not just image acquisition tools but also analytical instruments for quantitative measurements. Improving the quality assessment and quality control in both light and fluorescence microscopy is becoming a hot issue worldwide. The international standard for confocal microscopy measurements was developed by The International Organization for Standardization (ISO) in 2019 [6]. This standard provides guidelines for ensuring reproducible fluorescence confocal microscopy results. Additionally, a community-driven initiative, quality assessment, and reproducibility for instruments and images in light microscopy (QUAREP-LiMi) were established to improve the reproducibility of light microscopy image data [7].
Improving the quality control of microscopy measurements will support result comparisons between different instruments, different institutions, and ensure result reproducibility [8]. When microscopic data are obtained using two independent systems with different signal-detection efficiencies, directly comparing the resulting optical signal values is difficult. There is a need for a simple and widely applicable standardization procedure for benchmarking microscope systems and calibration using reference materials. Certified reference materials were employed to calibrate a fluorescence correlation spectroscopy measurement system [9], and this technique is useful for benchmarking the specifications of confocal microscope systems [10,11].
Standardization is essential for the accurate measurement of optical signals used in biological sample analysis in industrial fields. The optical signal value is used to quantitatively evaluate biological parameters in different applications, such as toxicity testing, environmental risk assessment, biomanufacturing, drug development, and regenerative medicine. The Organisation for Economic Co-operation and Development (OECD) guideline for toxicity testing using a cell-based assay with bioluminescence measurements was developed and widely used as an alternative to animal testing [12]. There is a critical need for high-quality optical signal measurements to improve the repeatability and intersystem comparability for luminescence and fluorescence analysis. It requires both proper optical signal measurement procedures and investigating deviations from the ideal proportionality between the optical signal and the measurement system readout. If a common “scale” (e.g., a reference LED light source) is applied for optical signal measurements, any signal emitted from samples can be compared within a laboratory, between manufacturer and manufacturer, between manufacturer and user, or between user and user, even if the data are taken by different systems [13].
Cell morphological feature analysis using light microscopy is widely used for drug discovery and industrial in-process control of cellular therapeutic products. However, characteristics of cell morphology obtained from microscopic images are frequently described in a subjective expression, such as “rounded shape” and “elongated shape.” Standardizing common descriptions, definitions, and mathematical formulae for quantification will both increase reliable data accumulation and provide a basis for assessing whether the data acquired by different people and systems can be quantitatively compared [14].
Standardization, including establishing “scale”s for measurement methods and an understanding of reliable measurement procedures, is becoming essential for industrial science and regulatory science fields, such as regenerative medicine and cellular therapeutics. Furthermore, even in the field of basic science, standardizing methods to improve reproducibility and intersystem comparability is important, especially in big data analysis. We would like to introduce metrology into bioluminescence, fluorescence measurement, and microscopy to make these systems calibrated, truly quantitative, and more reliable.
At the symposium session, the current situation of above topics is discussed. Niwa, K. and Ohmiya, Y. talk about optical signal measurements [3–5,12,13], Nomi, Y. introduces current standardization activity in the field of cell morphological measurement [14], Clayton, A. talks about quantitative single-molecule fluorescence measurements, and Halter, M. and Sasaki, A. talk about microscope benchmarking [9–11] and reference materials.
