Fluorescence calibration standards made from broadband emitters encapsulated in polymer beads for fluorescence microscop
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RESEARCH PAPER
Fluorescence calibration standards made from broadband emitters encapsulated in polymer beads for fluorescence microscopy and flow cytometry Katrin Hoffmann 1 & Nithiya Nirmalananthan-Budau 1 & Ute Resch-Genger 1 Received: 22 December 2019 / Revised: 31 March 2020 / Accepted: 15 April 2020 # The Author(s) 2020
Abstract We present here the design and characterization of a set of spectral calibration beads. These calibration beads are intended for the determination and regular control of the spectral characteristics of fluorescence microscopes and other fluorescence measuring devices for the readout of bead-based assays. This set consists of micrometer-sized polymer beads loaded with dyes from the liquid Calibration Kit Spectral Fluorescence Standards developed and certified by BAM for the wavelength-dependent determination of the spectral responsivity of fluorescence measuring devices like spectrofluorometers. To cover the wavelength region from 400 to 800 nm, two new near-infrared emissive dyes were included, which were spectroscopically characterized in solution and encapsulated in the beads. The resulting set of beads presents the first step towards a new platform of spectral calibration beads for the determination of the spectral characteristics of fluorescence instruments like fluorescence microscopes, FCM setups, and microtiter plate readers, thereby meeting the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas, e.g., medical diagnostics. This will eventually provide the basis for standardized calibration procedures for imaging systems as an alternative to microchannel slides containing dye solutions previously reported by us. Keywords Spectral calibration beads . Fluorescence microscopy . Dye . Polymer particle . Emission correction curve . Fluorescence standards
Introduction All fluorescence-based techniques generate signals that contain not only sample-related but also instrument-specific contributions. This limits the straightforward comparison of fluorescence data obtained, e.g., on the same instrument, but at different times, and particularly on different devices [1–7], and hampers quantification. Moreover, the recent trend to collect and provide reference data for all analytical techniques
Topical collection featuring Female Role Models in Analytical Chemistry. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02664-y) contains supplementary material, which is available to authorized users. * Ute Resch-Genger [email protected] 1
BAM Federal Institute for Materials Research and Testing, Richard-Willstaetter-Str. 11, 12489 Berlin, Germany
triggers the need for data bases that contain reliably corrected instrument-independent emission spectra of fluorescent reporters, probes, and sensors frequently used by the fluorescence community. In addition, fluorescence-based techniques like flow cytometry (FCM) and fluorescence microscopy applied in the life sciences and frequently also in
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