Fluorescence calibration standards made from broadband emitters encapsulated in polymer beads for fluorescence microscopy and flow cytometry

Katrin Hoffmann,Ute Resch-Genger,Nithiya Nirmalananthan-Budau

doi:10.1007/s00216-020-02664-y

Katrin Hoffmann, Ute Resch-Genger + Show 1 more

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https://doi.org/10.1007/s00216-020-02664-y

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Journal: Analusis	Publication Date: May 14, 2020
Citations: 7	License type: open-access

Affiliation: Federal Institute For Materials Research and Testing

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.Graphical abstract

Highlights

All fluorescence-based techniques generate signals that contain sample-related and instrument-specific contributions
To assess the spectral characteristics of fluorescence microscopes commonly used for fluorescence measurements from about 400 to 750 nm, only the standard dyes C, D, and E equaling the Bundesanstalt für Materialforschung und -prüfung (BAM)-certified dyes F003, F004, and F005 of the Kit Spectral Fluorescence standards were used in conjunction with the BAM software LinkCorrWin [7, 12, 33]
The dye solutions were previously assessed regarding a possible dependence of the shape of their emission spectra on typical excitation wavelengths used in fluorescence microscopy [33] and regarding their photostability under prolonged laser excitation

Summary

Introduction

All fluorescence-based techniques generate signals that contain sample-related and instrument-specific contributions. To rule out instrumentation as a major source of variability of emission data, easy to use, accessible, and generally accepted fluorescence standards are needed together with validated procedures for the control of instrument specifications and long-term performance [1, 8, 9] Such fluorescence standards, which can be physical devices like calibration lamps or chromophore-based chemical reference materials, should enable the determination of the relevant instrument parameters and instrument characteristics under application-relevant conditions as well as regular validation of instrument performance (IPV) to efficiently determine mandatory calibration intervals and to provide a regular basis for data quality [10]. Regular measurements of the intensity pattern of this standard can provide information on changes in the spectral characteristics of fluorescence measuring devices

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