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BAM Federal Institute for Materials Research and Testing

Standard Operation Procedure (SOP) for Use of

CERTIFIED REFERENCE MATERIALS

BAM-F001, BAM-F002a, BAM-F003, BAM-F004 and BAM-F005

Calibration Kit SPECTRAL FLUORESCENCE STANDARDS

for the Determination of the Relative Spectral Responsivity s(λ) of Fluorescence Instruments

1. Overview SCOPE AND LIMITATIONS

• Determination of the relative spectral responsivity s(λ) of fluorescence instruments. 1/s(λ) is termed emission correction curve. Both can be determined by LINKCORRWIN.

• Determination of corrected fluorescence emission spectra that are comparable across instruments.

• Characterization of the long-term stability of the emission channel of fluorescence instruments. Fluorescence spectra that are corrected accordingly do not contain contributions from aging of optical components in the emission channel.

For more information, see certificate.

KIT CONTENT

• 5 dyes BAM-F001–BAM-F005 covering the spectral region of 300 nm to 770 nm provided in portions of 5-15 μg in vials.

Dye Sigma Aldrich product no. BAM-F001 72594

BAM-F002a 07021 BAM-F003 96158 BAM-F004 74245 BAM-F005 94053

Kit complete 69336

• 100 ml of ethanol, Sigma Aldrich product number 34923 (see Safety Data Sheet) • BAM-certificate of the normalized corrected fluorescence emission spectra of the kit

dyes, see CD • BAM-Software LINKCORRWIN V.1.1.0.0., see CD • Certified corrected emission spectra of dye standards BAM-F001 - BAM-F005, readable by LINKCORRWIN STORAGE CONDITIONS, TESTED PROPERTIES, and MEASUREMENT CONDITIONS

Store Kit at 4 °C. Prepared working solutions must be used within 1 week. See certificate (enclosed).

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SCHEME 1: INSTRUCTIONS FOR USE (OVERVIEW)

Before you start: • Control the wavelength accuracy of the emission channel • Make sure that the detection system is operated within its linear range

Preparation of working solutions

100 ml ethanol • solvent • blank

10 ml 10 ml 10 ml 10 ml 10 ml

F001 F002a F003 F004 F005

Shake each closed vial and wait 15 min for complete dye dissolution

Similar measurement conditions for all Kit components with respect to • Measurement geometry • Optical components in emission channel (filter,

polarizer/polarizer settings etc.) • Detector settings (voltage etc.) • Spectral bandpass of emission monochromator/slit

width • Wavelength grid for CCDs • Integration time / scan speed • Sample temperature (data certified for 25±2 °C)

Choice of cells / cuvettes • Similar type for all Kit components • Properly closed to avoid uptake of water • Quarz cells recommended • Glass & polymer cells: wavelength region for use is

limited due to material absorption! • If a single cell is used for all measurements, then

rinse properly with ethanol between measurements

Similar measurement conditions as used for samples to be corrected recommended conditions

Dye-specific instrument settings • Emission range • Excitation wavelength • 3 - 10 scans as a rule of thumb

Fill solution of BAM-F001 into cell and measure, rinse cell, fill with ethanol, measure solvent spectrum or so-called blank.

Save spectra in any spread sheet program as ASCII, and transform into files with extension *.dat or *.csv

Repeat with F002a, F003, F004, and F005 and the corresponding blanks (blank spectra)

Keep dye solutions during calibration in properly closed cells: dark, (20-25) °C. Dye solutions can be stored in properly closed vials for max. 7 days: dark, 4 °C

Check statistical quality of data with LINKCORRWIN V.1.1.0.0.

If data are of insufficient statistical quality, repeat calibration with more scans

Emission correction curve 1/s(λ). The reciprocal of this curve yields s(λ).

Multiplication of measured spectra with 1/s(λ), or division by s(λ) yields spectrally corrected instrument-independent spectra.

dye λex / nm emission range / nm F001 280 300 – 450

F002a 315 330 – 530 F003 380 390 – 600 F004 420 450 – 700 F005 550 570 – 770

Averaging of data for each dye-solvent-pair in any spread sheet program

Special for CCD-Arrays: To guarantee the required identical wavelength grid for all emission standards, it is mandatory to measure the emission spectra of all the kit dyes within the same spectral range for the determination of the correction function. For F001-F005: 300-770 nm. For subsequent use of the correction curve, the spectra to be corrected always need to be measured within the same overall spectral range as the standards.

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SCHEMA 1: ARBEITSANWEISUNG (ÜBERSICHT)

Voraussetzungen: • Kontrolle der Wellenlängenrichtigkeit des Emissionskanals • Betrieb des Detektionssystems im linearen Bereich (vorher Linearität des Detektors bestimmen!)

Herstellung der Messlösungen

Identische Messbedingungen für alle Farbstoffe hinsichtlich • Messgeometrie • Optische Komponenten (Filter, Polarisator/Stellung etc.)

im Emissionskanal • Detektoreinstellungen (Spannung etc.) • Spektrale Spaltbreite des Emissionsmonochromators • Wellenlängenraster/Schrittweite für CCDs • Integrationszeit/Geschwindigkeit • Probentemperatur 25±2 °C

Anforderungen an Messzellen/Küvetten • Gleicher Typ • Fest verschlossen, um Wassereintrag zu vermeiden • Möglichst aus Quarz • Glas- & Polymerküvetten: Messbereich ist eingeschränkt

durch Absorption des Materials! • Wird dieselbe Küvette für alle Messungen verwendet,

dann gut reinigen mit Ethanol

Identische Mess-bedingungen wie zu korrigierende Proben/Spektren Empfohlene Messbedingungen

Emissionskorrekturkurve 1/s(λ). Bildung des Kehrwertes ergibt s(λ).

Multiplikation von gemessenen Spektren mit 1/s(λ), oder Division durch s(λ) ergibt spektral korrigierte, geräteunabhängige Spektren.

100 ml Ethanol • Lösungsmittel • Blindwert

10 ml 10 ml 10 ml 10 ml 10 ml

F001 F002a F003 F004 F005

Geschlossene Vials schütteln und 15 min warten, bis sich die Farbstoffe gelöst haben.

Spezifische Messbedingungen (siehe rechts) • Emissionsbereich • Anregungswellenlänge • 3 -10 Scans (Richtwert)

Messen von Farbstoff F001, Küvette reinigen, Messen des Lösungsmittelspektrums bzw. des Blindwertes.

Speichern der Spektren in einem spreadsheet Programm als ASCII Files; erforderliche Extension *.dat oder *.csv

Wiederholen mit F002a, F003, F004 und F005 und den entsprechenden Blindwerten

Lagerung der Farbstofflösungen während der Kalibrierung möglichst in einer fest verschlossenen Küvette: dunkel, (20–25) °C. Lagerung der Farbstofflösungen in fest verschlos-senen Gefäßen max. 7 Tage: dunkel, 4 °C

Prüfung der statistischen Qualität der Daten mit LINKCORRWIN V.1.1.0.0.

Bei unzureichender statistischer Qualität der Daten: Wiederholung mit mehr Scans

Farbstoff λex / nm EM-Spektralbereich / nm F001 280 300 – 450 F002a 315 330 – 530 F003 380 390 – 600 F004 420 450 – 700 F005 550 570 – 770

Mittelung der Daten für jedes Farbstoff-Lösungsmittel-Paar (Tabellenkalkulation)

Wichtig für CCD-Arrays: Um ein konstantes Wellen-längenraster für alle kalibrie-rungsrelevanten Standards zu erzeugen, ist es zwingend notwendig, alle für die Erstellung der Korrekturfunk-tion verwendeten Kit-Farbstoffe im gleichen Spektralbereich zu vermessen. F001-F005: 300-770 nm. Die Proben der zu korrigierenden Spektren müssen anschließend auch in dem für die Kalibrierung verwendeten Gesamtspektralbereich vermessen werden.

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2. Instruction for use 2.1. Kit handling

BEFORE YOU START: • Check the wavelength accuracy of the emission channel • Make sure that the instrument’s detection system is operated within its linear range

to avoid spectral distortions due to detector saturation (see also certificate).

SAMPLE PREPARATION • Dissolve each dye in 10 ml ethanol

MEASUREMENT CONDITIONS

• Measurement conditions are given in Scheme 1/Schema 1 and in the certificate • Set thermostate to 25 °C (±2 °C) • Use properly closed fluorescence cuvettes (quartz cells) to minimize water-uptake

and solvent evaporation, preferentially the same cuvette for all measurements. • Glass & polymer cells: wavelength region for use is limited due to material absorption! • All spectra need to be measured under identical conditions: spectral bandpass of the

emission monochromator, PMT voltage, scan speed (e.g. 120nm/min), step-size. • Only the parameters excitation wavelength, spectral bandpass of the excitation

monochromator and scanned spectral emission range can be varied amongst the kit dyes. These parameters have to be kept constant for each dye-blank-pair.

• Identical wavelength grids (“slits”) are required for the calculation of the global emission correction curve with LINKCORRWIN.

• To check the suitability of the chosen instrument settings, record a test spectrum in order to avoid saturation of the detection system. For this purpose, use of BAM-F003 is recommended.

Special for CCD-Arrays:

The emission spectra of F001 to F005 (or other dye combinations such as F002a to F004) always have to be measured with equidistant data points (wavelength scale) and wavelength grids as well as with equidistant wavelength intervals to ensure reliable data evaluation.

Dye λex / nm settings : calibration user samples F001- F005 280; 315; 380; 420; 550 300 - 770

NUMBER OF SCANS An emission correction curve of sufficient statistical quality can require several

emission scans and subsequent averaging to reduce noise. As a rule of thumb, the number of scans performed with the Kit dyes should exceed those run for conventional fluorescence measurements (identical instrument settings) by a factor of at least 3. We recommend to start with a number of 3-10 scans per dye.

BLANK CORRECTION

• The emission spectra of the solvent ethanol (blank) need to be measured under identical conditions as used for the Kit dyes. Both types of spectra will be employed for the determination of the emission correction curve with LINKCORRWIN.

• Start with BAM-F001 followed by blank spectrum BAM-F001 (only solvent, but using the conditions of dye F001), then dye F002a, blank spectrum F002a etc., eventually running several scans for each dye-solvent pair.

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Special for CCD-based Measurement Systems: It is very important to eliminate the excitation light (first and second order peaks) and the Raman band by subtracting the blank spectrum from the dye spectrum. This needs to be done before importing the data into LINKCORRWIN. Special care has to be taken with CCD-based systems, see Figure 1.

Figure 1a: Erroneous spectra Improper removal of scattered excitation light and Raman bands (see sharp peaks in Fig.1a) can lead to erroneous results when using such data with LINKCORRWIN. The most straightforward approach to avoid these uncertainities is the measurement of the Kit component spectra and the corresponding blanks using the complete wavelength region covered by Kit dyes, i.e. 300 to 770 nm, and to reduce the wavelength region for each dye to the special regions listed in the table of Scheme 1 (see Fig.1b).

Figure 1b: Reduced spectra

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2.2. Data handling and LINKCORRWIN DATA EXPORT AND EVALUATION The measured (averaged) emission spectra of the Kit components and respective blank spectra need to be saved in an ASCII file format for the calculation of the emission correction curve 1/s(λ) with LINKCORRWIN. The working principle of LINKCORRWIN is described in Chapter 3 and in the certificate. DO NOT normalize or smooth measured data. Make sure, that the ASCII-Files have the extension .dat or .csv, and do not contain extra signs, binary code elements or extra lines (no data) at the beginning or end of a file. Otherwise, manual editing of the ASCII-File is necessary (e.g. delete header information), so that only 2 data rows are left. LINKCORRWIN interprets the first data row as wavelength and the second as fluorescence intensity.

Figure 2: ASCII-file example: no header, only tab-separated columns

Figure 3a

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Make sure that for spectrally neighboring spectra, the wavelength scales/grids of both spectra are equidistant within their overlapping region (see Fig.3a/b)

Figure 3b LINKCORRWIN can handle two files per Kit component, i.e. one dye and one blank spectrum. If several scans are performed, the corresponding (dye and blank) spectra need to be averaged prior to use of LINKCORRWIN (either manually or by software).

If parts from the excitation light cannot be properly eliminated from the measured data via a blank correction, remove the corresponding peaks from the dye data. See also spectral data in Scheme 1.

USE OF LINKCORRWIN

IMPORTANT: There exist two versions of the BAM software LINKCORRWIN. Care must be taken to use LINKCORRWIN vers. 1.0.1.2. only in conjunction with BAM-F002 LINKCORRWIN vers. 1.1.0.0. only in conjunction with BAM-F002a

1. Insert CD and double click on LINKCORRWIN.EXE. Then choose the certificate file

BAM509Mx.CTF (x =1, 4 or 8; x: spectral bandpass of the emission monochromator M used for certification) that closely matches the spectral bandpass used for instrument calibration with BAM-F001 - BAM-F005 and click on it.

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2. Select your measured ASCII-files with OPEN SPEC from any directory of your computer

(local drives only!).

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3. After selection of the ASCII-files (dye spectra and blanks), the spectra are shown in the main window.

4. Click on button “Calculate” and the global emission correction curve appears. This curve,

shown in pink, equals the relative inverse spectral responsivity 1/s(λ) of the instrument to be calibrated (see also Figure 4).

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(Note: The „advanced options“ only have to be used in particular cases or scientific interpretations. They also provide a basis for further developments. Please use them only after consultation with BAM.) 5. The resulting emission correction curve is also statistically evaluated. A click on “Show

Log” opens the Logfile that lists all the activities of LINKCORRWIN (as *.txt).

6. Scrolling the Logfile reveals the results of a statistical test of the calibration data in the

peak region of the fluorescence spectra of BAM-F001 - BAM-F005. For data of insufficient statistical quality, LINKCORRWIN recommends, how often the fluorescence spectra should be remeasured for each dye and than averaged to obtain an improved correction curve with a better signal-to-noise ratio.

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7. Click on “Save result as”, create a name for the emission correction curve, and select a directory.

8. LINKCORRWIN creates a text-file and a log-file. The text-file equals the emission

correction curve 1/s(λλλλ) and consists of three columns, with the first column containing the wavelength values and the second one the calculated correction factors. The third column shows the uncertainty values.

9. Close LINKCORRWIN with click on “EXIT”.

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APPLICATION OF THE EMISSION CORRECTION CURVE Spectrally corrected emission spectra are obtained by multiplication of the measured blank corrected spectra with the overall emission correction curve 1/s(λ) (values in column “CF” in the text-file created by LINKCORRWIN using a spread-sheet program such as e.g. MICROCAL ORIGIN or MS EXCEL. Make sure that only values are multiplied that correspond to similar wavelengths. Figure 4: Determination of the relative spectral responsivity s(λ) of a fluorescence instrument with the Calibration Kit Spectral Fluorescence Standards and LINKCORRWIN. Bottom: Certified normalized corrected emission spectra (solid lines; (IC(λem)) of the Kit components and uncorrected, i.e. instrument-dependent emission spectra (dashed lines; IU(λem)), measured with the instrument to be calibrated. Middle: Individual quotients QF00x = IC(λem) / IU(λem), calculated with LINKCORRWIN for the Kit components. These quotients equal the inverse relative spectral responsivity of the instrument to be calibrated within the spectral region of the respective fluorescence standard or the individual emission correction curve. Top: Combined emission correction curve (solid pink line) calculated with LINKCORRWIN from the statistically weighted QF00x as well as s(λ) (black solid line).

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3. Theories of LINKCORRWIN WORKING PRINCIPLE

LINKCORRWIN was developed to link a series of wavelength-dependent quotients QF00x(λ) of corrected (IC(λ)) and measured, i.e. uncorrected (IU(λ)) fluorescence spectra of several spectral fluorescence standards, here BAM-F001 - BAM-F005, to a global correction curve covering the UV/vis/NIR spectral region in a defined fashion. The software can be used to link up to maximum six quotients. The global or combined correction curve equals the emission correction curve, i.e. the inverse relative spectral responsivity 1/s(λ) of the emission channel in the case of spectral emission standards and fluorescence emission spectra as is the case for BAM-F001 - BAM-F005. The procedure is illustrated in Figure 4.

PROCESSING of DATA

LINKCORRWIN can process 2 to 6 fluorescence spectra and the same number of blank, i.e. solvent spectra in ASCII-format (non-compelling header line; 2 columns for wavelength and fluorescence intensity values separated by tabulator or comma, floating format of numbers with decimal points or comma depending on separator-character; max 6 valid decades). Files must be named in 8.3 character format with extensions “DAT” or “CSV”. Each file can contain up to 3010 data pairs. The minimal supported wavelength resolution is 0.1 nm, the maximum 10 nm. The certified corrected fluorescence spectra of the BAM spectral fluorescence standards and the corresponding expanded relative uncertainties in 1nm data intervals are provided with the program as binary files or so-called certificate files, here BAM509Mx.CTF with x equalling 1, 4, and 8 (with the contributions of uncertainty). These files are in a format readable by LINKCORRWIN.

WINDOWS-FRAME-PROGRAM LINKCORRWINGUI

LINKCORRWIN GUI enables the use of the WINDOWS surface for processing of data, i.e. for instance path selection and file selection. All information is stored in the interface file LINKCORR.INI which controls the execution of LINKCORR.EXE. The kernel program does not contain interactive components. Thus, processing information or e.g. error messages are transferred from the output file LINKCORR.log to the frame program.

WORKING PRINCIPLE of LINKCORRWIN

1. Read-in of the data files: Some subroutines check the spectra to be loaded for ASCII-accordance of format, transform the format if necessary, sort the provided spectra according to wavelength and fluorescence standard-solvent or dye-blank pair automatically, and cut off all the data pairs the fluorescence intensities of which are below a threshold of 5% of the respective maximum value. Another routine assigns the measured uncorrected spectra to the corresponding BAM-certified spectra. Only if the program finds a wavelength accordance for the tails of all the measured dye and the accompanying blank spectra, the blank correction I( ) = Icust( ) - IBlank( ) is performed.

After loading the measured spectra in the assigned order, the certified spectra are loaded and interpolated for the same data intervals/wavelength step widths as used for the measured fluorescence spectra, if the data intervals of the corrected and uncorrected spectra differ. This interpolation is based on a third order polynomial regression of 5 data points of the certified spectra. The regression function is used to set values for the points to be interpolated in the wavelength grid of the user’s data. The signal-to-noise (S/N) ratio of the measured spectra in the intensity range of >97% of the maximum intensity are analyzed to obtain an estimate of the quality of the data provided by the user of BAM-F001-BAM-F005 and LINKCORRWIN. The routine employed applies a polynomial regression procedure, the highest order of which depends on the number of data points N, that lay within the range of the peak maximum of the measured

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fluorescence spectrum. The solutions of the polynomial equations ==

m

ixaxf i

i0

)(

for the parameters ai from m+1 down to 3 are represented by a matrix. A set of approximated polynomial model curves f(x) for the peak region is obtained. The deviations of the measured data points Ik from this polynomial curve of m’th power is expressed as χχχχ :

( )

mN

kk

N

kxfI

−=

−=1

2

)(χ

The polynomial curve that yields minimum deviation, i.e. a minimal value of χ is subsequently used for the calculation of the signal-to-noise ratio. In a first approximation, the reciprocal of χ provides a measure of the noise of the measured spectrum. As a rule of thumb, 1000 should be exceeded for a satisfying statistical quality of the measured data. If this criterion is not fulfilled, LINKCORRWIN recommends multiple recording of fluorescence spectra of the fluorescence standards to decrease the statistical uncertainty to a sufficient measure. Here, it should be taken into account, that the noise on an accordingly generated correction curve is transferred directly to every corrected fluorescence spectrum.

2. LINKCORRWIN searches for intersection points between the tails of subsequent, i.e. spectrally neighboring spectra, e.g. spectra of dyes BAM-F002a and BAM-F003, and also for the certified spectra. The intersection point between two spectra is the wavelength position at which the normalized fluorescence intensity values for the preceding dye curve (e.g. BAM-F002a) and the succeeding curve (e.g. BAM-F003) approach or cross each other. The 4-curves-intersection wavelength of the two spectrally neighboring uncorrected dye curves and the two corresponding certified dye spectra is the data pair ( ,I) within the wavelength grid closest to the average of the two intersection wavelengths. This procedure is repeated for all intersection regions.

3. At both sides of the intersection wavelengths, a wavelength range is fixed for which so-called connecting factors i are calculated according to a statistical weighting procedure described by J. A. Gardecki and M. Maroncelli, Appl. Spectrosc. 1998, 52,1179-89.

Ii( ) , Ij( ) : intensities of the succeeding BAM-certified corrected spectra I, j at Ji( ) , Jj( ) : intensities of the succeeding measured uncorrected spectra i , j at CFi( ), CFj( ) : quotients [I i ( )] / [Ji ( )] and [I j ( )] / [Jj ( )]

j : connecting factors for quotient at transition i j i : cumulative factors

CF ( ) : linked, statistically weighted quotients equaling global correction curve.

=

λ

λ

λσ

λσα

λ

λ

)(1

)()(

)(

,

2

,

2

mji

mjimj

mi

j

CFCF

with +=

)(

)(

)(

1

)(

12

)(2

,

mj

mi

ji CF

CF

JJmji

λ

λ

λλσ λ

∏=

=i

ki k

1αβ

15

=

==N

im

N

immiiCF

CF m

1

2

1

2

)(1

)()(

)(λσ

λσλβλ with

[ ])(

2

)()(2

λλβ

λσmi

m JCF mii=

From the first connecting factor 1 1.0 follows, that the overall correction curve is identically to the first quotient [I 1 ( )] / [J1 ( )] up to the wavelength position of the first intersection. LINKCORRWIN calculates the second connecting factor 2 within a wavelength region of ca. ± 8 nm at both sides of the first intersection wavelength if the default values are used. If desired, the user can define spectral ranges via the option line of the LINKCORRWIN -controlling file LINKCORR.INI. In contrast to Gardecki and Maroncelli, we decided to restrict this spectral range, since at the tail regions of fluorescence spectra, we sometimes observed bending of the quotients [I i ( )] / [Ji ( )] in the direction of smaller values of I i and / or Ji. To avoid the introduction of systematic errors within the linking procedure, LINKCORRWIN uses a smaller wavelength range to obtain an j that is more adequately weighted statistically. After completion of the calculation of the j’s and the cumulative product ß, the global correction curve is calculated. In contrast to the procedure of Gardecki and Maroncelli, LINKCORRWIN does not employ the equation for CF(λ) within the full range. Using the default mode of the program, the averaging function of this equation is restricted to the values at the intersection wavelengths. For all other spectral regions or parts of the single quotients CFi(λ), no averaging is performed, but the factor ßi is exclusively applied for all the points between the intersection wavelengths. With the option “k”, LINKCORRWIN averages all the values of the overlapping spectra for the same spectral range for which the ’js have been previously calculated.

We chose the described weighting procedure deviating from the Marconelli approach as we often found small shoulders or jumps within the intersection wavelength ranges where the correction curve is calculated from a single quotient CFi(λ) and where two quotients CFi(λ) and CFi+1(λ) are averaged. This is due to increasingly different slopes of the single quotients CFi(λ) with increasing distance from the intersection wavelength. To avoid such jumps or steps in the global emission correction curve, the spectral window in which weighting by LINKCORRWIN in the default mode is performed is therefore reduced to the single value at the intersection wavelength. The values at wavelengths preceding or succeeding the intersection wavelength are treated by unweighted coefficients ßI or ßI+1 .

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ADDITIONAL INFORMATION

Hollandt, J.; Taubert, R. D.; Seidel, J.; Resch-Genger, U.; Gugg-Helminger, A.; Pfeifer, D; Monte,, C.; "Traceability in Fluorometry: Part I Physical Standards", J. Fluoresc. 2005, 15, 311. Resch-Genger, U.; Pfeifer, D.; Pilz, W.; Monte,, C.; Hoffmann, A.; Spieles, M.; Rurack, K.; Hollandt, J.; Taubert, D.; Schönenberger, B.; Nording, P. "Traceability in Fluorometry: Part II Spectral Fluorescence Standards", J. Fluoresc. 2005, 15, 325. Resch-Genger, U.; Hoffmann, K.; Nietfeld, W.; Engel, A.; Neukammer, J.; Nitschke, R.; Ebert, B.; Macdonald, R. "How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards", J. Fluoresc. 2005, 15, 347. Hoffmann, K.; Resch-Genger, U.; Nitschke, R. "Simple Tool for the Standardization of Confocal Spectral Imaging Systems", G.I.T Imaging & Microscopy 2005, 3, 18. Monte, C.; Pilz, W.; Resch-Genger, U. "Linking Fluorescence Spectroscopy to the Scale of Spectral Sensitivity: the BAM Reference Fluorometer" in "Optical Diagnostics", Hansen, M. L., Farrell, P. V. (eds.), Proc. SPIE 2005, 5880, 588019-1. Hoffmann, K.; Monte, C.; Pfeifer, D.; Resch-Genger, U. "Standards in Fluorescence Spectroscopy: Simple Tool for the Characterization of Fluorescence Instruments", G.I.T Laboratory Journal 2005, 6, 29. Monte, C.; Resch-Genger, U.; Pfeifer, D.; Taubert, D. R.; Hollandt, J. "Linking Fluorescence Measurements to Radiometric Units", Metrologia 2006, 16, S89. Monte, C.; Hoffmann, K.; Pfeifer, D.; Hoffmann, A.; Resch-Genger, U. "The Kit Spectral Fluorescence Standards: A Simple Calibration Tool for the Standardization of the Spectral Characteristics of Fluorescence Instruments", J. Fluoresc. 2006, 16, 441.

CONTACT / Technical Support

Dr. U. Resch-Genger Phone: ++49(0)30-8104-1134 Dipl.-Ing. A. Hoffmann Phone: ++49(0)30-8104-5879 AG Biophotonik BAM Federal Institute for Materials Research and Testing Richard-Willstaetter-Str. 11 D-12489 Berlin Germany Fax: ++49(0)30-8104-1157 Email: ute.resch@bam.de Email: angelika.hoffmann@bam.de Email: dietmar.pfeifer@bam.de