Comparability of Fluorescence Microscopy Data and Need for Instrument Characterization of Spectral Scanning Microscopes

Hoffmann, Katrin; Resch‐Genger, Ute; Nitschke, Roland

doi:10.1007/4243_2008_028

Cited by 8 publications

(19 citation statements)

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“…With the increase in quantitative imaging and in medical applications of fluorescence based techniques regular performance checks with standardised samples and defined acquisition protocols will be key to ensure that appropriate experimental data are acquired [6,10]. Efforts are being made to develop suitable calibration and validation standards required to standardise microscopic imaging [6].…”

Section: Resultsmentioning

confidence: 99%

“…Despite their popularity and wide spread use there are very few if any tools provided by the manufacturers themselves to monitor confocal performance following the purchase of a new system. It might just be a slide with a fluorescently labelled section of plant tissue (Convallaria spec., ‘lily of the valley’) which provides ‘pretty’ multi-colour images but the use of these biological samples as test specimens for the evaluation of a confocal microscope is very limited [6,7]. …”

Section: Introductionmentioning

confidence: 99%

“…Due to the increasing demand for quantitative analysis of biological samples and the complexity of biological research with experiments stretching over many months or years or even spread over several laboratories it is essential to ensure the accuracy and comparability of the data recorded on a confocal microscope [6,8]. When performing quantitative fluorescence imaging the main source of concern would be temporal and spatial signal intensity variations introduced by the instrument itself (which are by no means specific to the confocal microscope) [8,9,10].…”

Section: Introductionmentioning

confidence: 99%

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ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

Hng

Dormann

2013

PLoS ONE

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Laser scanning confocal microscopy has become an invaluable tool in biomedical research but regular quality testing is vital to maintain the system’s performance for diagnostic and research purposes.Although many methods have been devised over the years to characterise specific aspects of a confocal microscope like measuring the optical point spread function or the field illumination, only very few analysis tools are available. Our aim was to develop a comprehensive quality assurance framework ranging from image acquisition to automated analysis and documentation. We created standardised test data to assess the performance of the lasers, the objective lenses and other key components required for optimum confocal operation.The ConfocalCheck software presented here analyses the data fully automatically. It creates numerous visual outputs indicating potential issues requiring further investigation. By storing results in a web browser compatible file format the software greatly simplifies record keeping allowing the operator to quickly compare old and new data and to spot developing trends.We demonstrate that the systematic monitoring of confocal performance is essential in a core facility environment and how the quantitative measurements obtained can be used for the detailed characterisation of system components as well as for comparisons across multiple instruments.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

Hng

Dormann

2013

PLoS ONE

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“…The transfer and adaptation of evaluated and established procedures and standards for instrument characterization and instrument performance validation from one fluorescence technique to another requires proper consideration of method-inherent requirements on standards, and of scope-specific limitations of methods and standards [6,50,131]. This includes, for instance, adaptation of measurement parameters, measurement geometry, sample or standard format, excitation wavelength(s), and (photochemical and thermal) stability [49,50].…”

Section: Adaptation Of Fluorescence Standards To Different Fluorescenmentioning

confidence: 99%

“…This includes, for instance, adaptation of measurement parameters, measurement geometry, sample or standard format, excitation wavelength(s), and (photochemical and thermal) stability [49,50]. The latter is of special importance for techniques using lasers as excitation sources with their strongly enhanced excitation intensity or spectral radiance and fixed excitation wavelength [131]. Also, the standard's luminescence lifetime can be critical as this parameter controls the standard's suitability for techniques that use pulsed excitation light sources, or that employ short measurement or integration times (pixel times) such as fluorescence microscopy [6,131].…”

Section: Adaptation Of Fluorescence Standards To Different Fluorescenmentioning

confidence: 99%

Fluorescence standards: Classification, terminology, and recommendations on their selection, use, and production (IUPAC Technical Report)

Resch‐Genger

DeRose

2010

Pure and Applied Chemistry

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Chromophore-based fluorescence standards for the characterization of photoluminescence measuring systems and the determination of relevant fluorometric quantities are classified according to their scope and area of application. General and type-specific requirements for suitable standards are derived for each class of standards. Metrological requirements linked to the realization of comparable measurements are addressed and recommendations on selecting, using, and developing fluorescence standards are given.

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Measurement of wheat germ agglutinin binding with a fluorescence microscope

2009

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Signal intensity in fluorescence microscopy is often measured relative to arbitrary standards. We propose a calibration method based on a solution of the same fluorophore, whose binding to cells needs to be quantified. The method utilizes the low sensitivity of intensity to the object distance in wide-field imaging of uniform materials. Liquid layers of slowly varying depth were prepared by immersing a spherical lens into a drop of a fluorophore placed on a slide. Flatfield-corrected images of the contact and surrounding areas showed linear dependence of the gray level on the depth of fluorescent liquid. This allowed conversion of the measured intensity into the number of molecules per unit area. The method was applied to different cell types stained by WGA-Alexa 488 and WGA-TRITC. Consistent results were obtained by comparing microscopy with flow cytometry, comparing imaging through different objectives and comparing different WGA conjugates. Reproducibility of calibration was within 97% when low magnification was used. Fluorescence of free and bound WGA was found to be different, however, and therefore precise measurement of the number of cell-bound molecules was problematic in this particular system. We conclude that the method achieves reliable measurement of cellular staining in the units of soluble fluorophore. For probes whose fluorescent properties are unaffected by binding, quantification of staining in true molecular units should be possible. 1-4). Conceptually, the first step in this type of measurements is to compare on a calibrated spectrofluorimeter the brightness of a bead suspension to the brightness of a standard solution of a fluorochrome. This allows equating the brightness of one bead to the brightness of a certain number of fluorochrome molecules, and that number is designated as the MESF value of a bead. Commercial beads with specified MESF values are available and are used to calibrate flow cytometers for analyzing unknown cells or particles. Measurements of antigen expression (4-6), ligand-receptor interactions, (7-9) and telomere length (10,11) have proved the usefulness of standardized units of fluorescence intensity.Fluorescence microscopy shares many applications with flow cytometry, but in contrast with the latter it lacks convenient standards that would allow quantification in absolute units. Due to the ever increasing importance of digital imaging in medicine and drug discovery, the need for standardization in microscopy has been repeatedly stressed by many authors (12-15). Although a number of different approaches to calibrate fluorescence microscopes have been proposed (15-17), most of them rely on more or less arbitrarily chosen units of intensity.

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Comparability of Fluorescence Microscopy Data and Need for Instrument Characterization of Spectral Scanning Microscopes

Cited by 8 publications

References 50 publications

ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

Fluorescence standards: Classification, terminology, and recommendations on their selection, use, and production (IUPAC Technical Report)

Measurement of wheat germ agglutinin binding with a fluorescence microscope

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