Fluorescence performance standards for confocal microscopy (original) (raw)
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Cytometry Part A, 2014
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.
Characterization and Calibration in Wide Field and Sectioned Fluorescence Microscopy SIPcharts
Springer Series on Fluorescence
In the booming fields of the life and material sciences, advances are taking place on all fronts and often involve the use of luminescence techniques as analytical tools and detection methods due to their high sensitivity, intrinsic selectivity, noninvasive (or at least minimally invasive) character, comparative ease of use, potential for multiplexing applications, and remote accessibility of signals. Despite the fact that the measurement of fluorescence-with its birth marked by the study of Sir Stokes on quinine sulfate in 1852-is not a new technique and many fluorescence techniques have matured to a state where quantification is desired, standardization of the broad variety of fluorescence methods and applications is still in its infancy as compared to other prominent (bio)analytical methods. It is still often overlooked that all types of fluorescence measurements yield signals containing both analyte-specific and instrument-specific contributions. Furthermore, the absorption and fluorescence of most fluorophores is sensitive to their microenvironment, and this can hamper quantification based on measurements of relative fluorescence intensities as well as accurate measurements of absolute fluorescence intensities. Hence, the realization of a truly quantitative measurement is inherently challenging. This situation renders quality assurance in fluorometry very important, especially with respect to the increasing complexity of instrumentation, and the blackbox-type of presentday instruments and software. This may compromise future applications of fluorescence techniques in strongly regulated areas like medical diagnostics and clinical chemistry that are within reach. As a result, there is an ever increasing need for (a) recommendations and guidelines for the characterization and performance validation of fluorescence instrumentation and the performance of typical fluorescence measurements, and (b) for an improved understanding of fluorescence-inherent sources of error. This is closely linked to the availability of suitable and easily handled standards that can be operated under routine analytical conditions, are adequately characterized, and meet overall accepted quality criteria. Within this context, the aim of this book is to provide a unique overview on the current state of instrumentation and application of a very broad variety of fluorescence techniques employed in the material and especially in the life sciences thereby highlighting the present state of quality assurance and the need VIII Preface for future standards. Methods included span microfluorometric techniques used for immunoassays, fluorescence microscopic and imaging techniques including single molecule spectroscopy, flow cytometry and fluorescence in situ hybridization to the microarray technology and technologies used in biomedical diagnostics like in vivo fluorescence imaging. Method-inherent advantages, limitations, and sources of uncertainties are addressed, often within the context of typical and upcoming applications. The ultimate goal is to make users of fluorescence techniques more aware of necessary steps to improve the overall reliability and comparability of fluorescence data to encourage the further broadening of fluorescence applications. I wish to express my appreciation and special thanks to the individuals who insisted and encouraged me in the preparation of this book. These include Dr.
An automated protocol for performance benchmarking a widefield fluorescence microscope
Cytometry Part A, 2014
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.
The Development of Fluorescence Intensity Standards
2001
The use of fluorescence as an analytical technique has been growing over the last 20 years. A major factor in inhibiting more rapid growth has been the inability to make comparable fluorescence intensity measurements across laboratories. NIST recognizes the need to develop and provide primary fluorescence intensity standard (FIS) reference materials to the scientific and technical communities involved in these assays. The critical component of the effort will be the cooperation between the Federal laboratories, the manufacturers, and the technical personnel who will use the fluorescence intensity standards. We realize that the development and use of FIS will have to overcome many difficulties. However, as we outline in this article, the development of FIS is feasible.
Fluorophores for Confocal Microscopy: Photophysics and Photochemistry
Fluorescence is probably the most important optical readout mode in biological confocal microscopy because it can be much more sensitive and specific than absorbance or reflectance, and because it works well with epi-illumination, which greatly simplifies scanner design. These advantages of fluorescence are critically dependent on suitable fluorophores that can be tagged onto biological macromolecules to show their location, or whose optical properties are sensitive to the local environment. Despite the pivotal importance of good fluorophores, little is known about how rationally to design good ones. Whereas the concept of confocal microscopy is only a few decades old and nearly all the optical, electronic, and computer components to support it have been developed or redesigned in the last few years, the most popular fluorophores were developed more than a century ago (in the case of fluoresceins or rhodamines) or several billion years ago [in the case of phycobiliproteins and green fluorescent proteins (GFPs)].
IEEE Transactions on Medical Imaging, 2016
To date, no emerging preclinical or clinical near-infrared fluorescence (NIRF) imaging devices for non-invasive and/or surgical guidance have their performances validated on working standards with SI units of radiance that enable comparison or quantitative quality assurance. In this work, we developed and deployed a methodology to calibrate a stable, solid phantom for emission radiance with units of mW • sr −1 • cm −2 for use in characterizing the measurement sensitivity of ICCD and IsCMOS detection, signal-to-noise ratio, and contrast. In addition, at calibrated radiances, we assess transverse and lateral resolution of ICCD and IsCMOS camera systems. The methodology allowed determination of superior SNR of the ICCD over the IsCMOS camera system and superior resolution of the IsCMOS over the ICCD camera system. Contrast depended upon the camera settings (binning and integration time) and gain of intensifier. Finally, because of architecture of CMOS and CCD camera systems resulting in vastly different performance, we comment on the utility of these systems for small animal imaging as well as clinical applications for non-invasive and surgical guidance.
Recommendations for Fluorescence Instrument Qualification: The New ASTM Standard Guide
Analytical Chemistry, 2010
Aimed at improving quality assurance and quantitation for modern fluorescence techniques, ASTM International (ASTM) is about to release a Standard Guide for Fluorescence, reviewed here. The guide's main focus is on steady state fluorometry, for which available standards and instrument characterization procedures are discussed along with their purpose, suitability, and general instructions for use. These include the most relevant instrument properties needing qualification, such as linearity and spectral responsivity of the detection system, spectral irradiance reaching the sample, wavelength accuracy, sensitivity or limit of detection for an analyte, and day-today performance verification. With proper consideration of method-inherent requirements and limitations, many of these procedures and standards can be adapted to other fluorescence techniques. In addition, procedures for the determination of other relevant fluorometric quantities including fluorescence quantum yields and fluorescence lifetimes are briefly introduced. The guide is a clear and concise reference geared for users of fluorescence instrumentation at all levels of experience and is intended to aid in the ongoing standardization of fluorescence measurements. Recent developments in quantitative fluorescence-based assays in clinical, pharmaceutical, biotechnological, and other areas, in conjunction with global trends to harmonize measurements, traceability, and accreditation, 1,2 have spurred the demand for fluorescence standards and related standardization documents. The latter include carefully evaluated standard operating procedures, guidelines, and recommendations for instrument characterization and performance verification. Fluorescence standards include physical standards, e.g., a calibrated light source, and chemical standards, such as solid or liquid reference materials. Suitable examples should be robust, easy-to-use, readily available and, when appropriate, given with values that are SI-traceable,