6 research outputs found
Narrow-Band Emitting Solid Fluorescence Reference Standard with Certified Intensity Pattern
The
development of a lanthanum-phosphate glass doped with several
rare-earth-ions for use as solid fluorescence standard is described.
The cuvette-shaped reference material which shows a characteristic
emission intensity pattern upon excitation at 365 nm consisting of
a multitude of relatively narrow emission bands in the wavelength
region between 450 and 700 nm is intended for the day-to-day performance
validation of fluorescence measuring devices. Evaluation of the fluorescent
glass includes the determination of all properties which can affect
its relative emission intensity profile or contribute to the uncertainty
of the certified values like absorption spectra, fluorescence anisotropy,
excitation wavelength, and temperature dependence of the spectroscopic
features, homogeneity of fluorophore distribution, and photo- and
long-term stability. Moreover, a certification procedure was developed
including the normalization of the intensity profile consisting of
several narrow emission bands and the calculation of wavelength-dependent
uncertainties. Criteria for the design, characterization, and working
principle of the new reference material BAM-F012 are presented, and
possible applications of this ready-to-use fluorescence standard are
discussed
Integrating Sphere Setup for the Traceable Measurement of Absolute Photoluminescence Quantum Yields in the Near Infrared
There is an increasing interest in chromophores absorbing
and emitting
in the near-infrared (NIR) spectral region, e.g., for applications
as fluorescent reporters for optical imaging techniques and hence,
in reliable methods for the characterization of their signal-relevant
properties like the fluorescence quantum yield (Φ<sub>f</sub>) and brightness. The lack of well established Φ<sub>f</sub> standards for the NIR region in conjunction with the need for accurate
Φ<sub>f</sub> measurements in transparent and scattering media
encouraged us to built up an integrating sphere setup for spectrally
resolved measurements of absolute fluorescence traceable to radiometric
scales. Here, we present the design of this setup and its characterization
and validation including an uncertainty budget for the determination
of absolute Φ<sub>f</sub> in the visible and NIR. To provide
the basis for better measurements of Φ<sub>f</sub> in the spectral
window from ca. 600 to 1000 nm used, e.g., for optical imaging, the
absolute Φ<sub>f</sub> of a set of NIR chromophores covering
this spectral region are measured and compared to relative values
obtained using rhodamine 101 as Φ<sub>f</sub> standard. Additionally,
the absolute Φ<sub>f</sub> values of some red dyes that are
among the most commonly used labels in the life sciences are presented
as well as the absolute quantum yield of an optical probe for tumor
imaging
State-of-the Art Comparability of Corrected Emission Spectra. 2. Field Laboratory Assessment of Calibration Performance Using Spectral Fluorescence Standards
In the second part of this two-part series on the state-of-the-art
comparability of corrected emission spectra, we have extended this
assessment to the broader community of fluorescence spectroscopists
by involving 12 field laboratories that were randomly selected on
the basis of their fluorescence measuring equipment. These laboratories
performed a reference material (RM)-based fluorometer calibration
with commercially available spectral fluorescence standards following
a standard operating procedure that involved routine measurement conditions
and the data evaluation software LINKCORR developed and provided by
the Federal Institute for Materials Research and Testing (BAM). This
instrument-specific emission correction curve was subsequently used
for the determination of the corrected emission spectra of three test
dyes, X, QS, and Y, revealing an average accuracy of 6.8% for the
corrected emission spectra. This compares well with the relative standard
uncertainties of 4.2% for physical standard-based spectral corrections
demonstrated in the first part of this study (previous paper in this
issue) involving an international group of four expert laboratories.
The excellent comparability of the measurements of the field laboratories
also demonstrates the effectiveness of RM-based correction procedures
State-of-the Art Comparability of Corrected Emission Spectra.1. Spectral Correction with Physical Transfer Standards and Spectral Fluorescence Standards by Expert Laboratories
The development of fluorescence applications in the life
and material
sciences has proceeded largely without sufficient concern for the
measurement uncertainties related to the characterization of fluorescence
instruments. In this first part of a two-part series on the state-of-the-art
comparability of corrected emission spectra, four National Metrology
Institutes active in high-precision steady-state fluorometry performed
a first comparison of fluorescence measurement capabilities by evaluating
physical transfer standard (PTS)-based and reference material (RM)-based
calibration methods. To identify achievable comparability and sources
of error in instrument calibration, the emission spectra of three
test dyes in the wavelength region from 300 to 770 nm were corrected
and compared using both calibration methods. The results, obtained
for typical spectrofluorometric (0°/90° transmitting) and
colorimetric (45°/0° front-face) measurement geometries,
demonstrated a comparability of corrected emission spectra within
a relative standard uncertainty of 4.2% for PTS- and 2.4% for RM-based
spectral correction when measurements and calibrations were performed
under identical conditions. Moreover, the emission spectra of RMs
F001 to F005, certified by BAM, Federal Institute for Materials Research
and Testing, were confirmed. These RMs were subsequently used for
the assessment of the comparability of RM-based corrected emission
spectra of field laboratories using common commercial spectrofluorometers
and routine measurement conditions in part 2 of this series (subsequent
paper in this issue)
State-of-the Art Comparability of Corrected Emission Spectra.1. Spectral Correction with Physical Transfer Standards and Spectral Fluorescence Standards by Expert Laboratories
The development of fluorescence applications in the life
and material
sciences has proceeded largely without sufficient concern for the
measurement uncertainties related to the characterization of fluorescence
instruments. In this first part of a two-part series on the state-of-the-art
comparability of corrected emission spectra, four National Metrology
Institutes active in high-precision steady-state fluorometry performed
a first comparison of fluorescence measurement capabilities by evaluating
physical transfer standard (PTS)-based and reference material (RM)-based
calibration methods. To identify achievable comparability and sources
of error in instrument calibration, the emission spectra of three
test dyes in the wavelength region from 300 to 770 nm were corrected
and compared using both calibration methods. The results, obtained
for typical spectrofluorometric (0°/90° transmitting) and
colorimetric (45°/0° front-face) measurement geometries,
demonstrated a comparability of corrected emission spectra within
a relative standard uncertainty of 4.2% for PTS- and 2.4% for RM-based
spectral correction when measurements and calibrations were performed
under identical conditions. Moreover, the emission spectra of RMs
F001 to F005, certified by BAM, Federal Institute for Materials Research
and Testing, were confirmed. These RMs were subsequently used for
the assessment of the comparability of RM-based corrected emission
spectra of field laboratories using common commercial spectrofluorometers
and routine measurement conditions in part 2 of this series (subsequent
paper in this issue)
State-of-the Art Comparability of Corrected Emission Spectra.1. Spectral Correction with Physical Transfer Standards and Spectral Fluorescence Standards by Expert Laboratories
The development of fluorescence applications in the life
and material
sciences has proceeded largely without sufficient concern for the
measurement uncertainties related to the characterization of fluorescence
instruments. In this first part of a two-part series on the state-of-the-art
comparability of corrected emission spectra, four National Metrology
Institutes active in high-precision steady-state fluorometry performed
a first comparison of fluorescence measurement capabilities by evaluating
physical transfer standard (PTS)-based and reference material (RM)-based
calibration methods. To identify achievable comparability and sources
of error in instrument calibration, the emission spectra of three
test dyes in the wavelength region from 300 to 770 nm were corrected
and compared using both calibration methods. The results, obtained
for typical spectrofluorometric (0°/90° transmitting) and
colorimetric (45°/0° front-face) measurement geometries,
demonstrated a comparability of corrected emission spectra within
a relative standard uncertainty of 4.2% for PTS- and 2.4% for RM-based
spectral correction when measurements and calibrations were performed
under identical conditions. Moreover, the emission spectra of RMs
F001 to F005, certified by BAM, Federal Institute for Materials Research
and Testing, were confirmed. These RMs were subsequently used for
the assessment of the comparability of RM-based corrected emission
spectra of field laboratories using common commercial spectrofluorometers
and routine measurement conditions in part 2 of this series (subsequent
paper in this issue)