Method and kit for calibrating a photoluminescence measurement
View Patent ↗The invention is directed to a method and a kit for calibrating a photoluminescence measurement system, in particular a fluorescence measurement system. The kit includes a number of fluorescence standards i and their corrected and certified fluorescence spectra I i (λ), whereby the fluorescence standards i are selected, so that their spectrally corrected fluorescence spectra I i (λ) cover a broad spectral range with high intensity. The standards are characterized by large half-widths FWHM i of their bands of at least 1400 cm −1 . According to the method of the invention, partial correction functions F i (λ) are generated by forming the quotient of the measured fluorescence spectra J i (λ) and the corresponding corrected fluorescence spectra I i (λ), which are then combined to form a total correction function F(λ) for a broad spectral range. The combination factors α i are hereby computed by statistical averaging of consecutive partial correction functions F i (λ) over only a predefined, limited overlap region λ i/i+1 ±Δλ OL about the mutual crossover wavelength λ i/i+1 .
1. A method for calibrating a photoluminescence measurement system, in particular a fluorescence measurement system, the method comprising:
(a) measuring fluorescence spectra (J i (λ)) of a plurality of fluorescence standards (i) with the photoluminescence measurement system to be calibrated, wherein the fluorescence standards (i) are selected, so that their combined spectrally corrected and normalized fluorescence spectra (I i (λ)) cover a predefined spectral range in such a way, that the fluorescence bands of the spectrally corrected and normalized fluorescence spectra (I i (λ)) have at least a minimum predefined intensity at mutual crossover wavelengths (λ i/i+1 ), wherein the spectrally corrected and normalized fluorescence spectra (I i (λ)) is sequentially arranged;
(b) calculating combination factors (α i ) through statistical averaging of consecutive partial correction functions (F i (λ)), wherein the partial correction functions (F i (λ)) are formed by forming the quotient of the measured fluorescence spectra (J i (λ)) and the corresponding spectrally corrected and normalized fluorescence spectra (I i (λ)) of the fluorescence standards (i), and wherein the statistical averaging takes place over a predefined, limited overlap region (λ i/i+1 ±Δλ OL ) about the mutual crossover wavelengths (λ i/i+1 ); and
(c) determining a total correction function (F(λ)), for the predefined spectral range by combination of the partial correction functions (F i (λ)), which are weighted according to the combination factors (α i ).
2. The method of claim 1 , wherein the spectral overlap region (λ i/i+1 ±Δλ OL ; OL=overlap), where the combination factors (α i ) are calculated, corresponds to a range of at most ±12 nm about the mutual crossover wavelengths (λ i/i+1 ).
3. The method of claim 1 , wherein the functional dependence of the total correction function (F(λ)) is calculated in a predefined, limited combination region (λ i/i+1 ±Δλ LK ; LK=linkage) about the mutual crossover wavelengths (λ i/i+1 ) through statistical averaging of the weighted, consecutive partial correction functions (F i (λ)).
4. The method according to claim 3 , wherein the functional dependence of the total correction function (F(λ)) outside the predefined combination region (λ i/i+1 ±Δλ LK ) about the mutual crossover wavelengths (λ i/i+1 ) corresponds to the functional dependence of the weighted, un-averaged partial correction functions (F i (λ)).
5. The method according to claim 3 , wherein the predefined combination region (λ i/i+1 ±Δλ LK ) about the crossover wavelengths (λ i/i+1 ) includes ±5 nm about the corresponding mutual crossover wavelengths (λ i/i+1 ).
6. The method according to claim 1 , wherein the predefined spectral range of the total correction function (F(λ)) covers at least in part the UV/vis/NIR spectral range.
7. The method according to claim 1 , wherein the predefined spectral range of a total correction function (F(λ)) for correcting the emission extends from 310 to 730 nm and for correcting the excitation at least from 250 to 630 nm.
8. The method according to claim 1 , wherein the predefined minimum intensity of the fluorescence bands of the spectrally corrected and normalized fluorescence spectra (I i (λ)) at the mutual crossover wavelengths (λ i/i+1 ) in the UV/vis/NIR spectral range with λ≦700 nm is at least 20% of a maximum intensity of mutually overlapping flanking bands.
9. The method according to claim 1 , wherein the predefined minimum intensity of the fluorescence bands of the spectrally corrected and normalized fluorescence spectra (I i (λ)) at the mutual crossover wavelengths (λ i/i+1 ) in the NIR spectral range with λ>700 nm is at least 10% of a maximum intensity of a mutually overlapping flanking bands.
10. The method according to claim 1 , wherein intensities of the spectrally corrected and normalized fluorescence spectra (I i (λ)) within the spectral overlap region (λ i/i+1 ±Δλ OL ), where the combination factors (α i ) are calculated, in the UV/vis spectral range with λ≦700 nm are at least 10% in relation to maximum intensities of mutually overlapping flanking bands.
11. The method according to claim 1 , wherein the partial correction functions (F i (λ)) are calculated by dividing the spectrally corrected and normalized fluorescence spectra (I i (λ)) by the corresponding measured fluorescence spectra (J i (λ)), and a spectrum corrected for a blank value is corrected by wavelength-correlated multiplication with the total correction function (F(λ)).
12. The method according to claim 1 , wherein a wavelength-dependent combined uncertainty for the predefined spectral range of total correction function (F(λ)) is computed from respective combined uncertainties for individual fluorophores.
13. A method for calibrating a photoluminescence measurement system, in particular a fluorescence measurement system, the method comprising:
(a) measuring fluorescence spectra (J i (λ)) of a plurality of fluorescence standards (i) with the photoluminescence measurement system to be calibrated, wherein the fluorescence standards (i) are selected, so that their combined spectrally corrected and normalized fluorescence spectra (I i (λ)) cover a predefined spectral range in such a way, that the fluorescence bands of the spectrally corrected and normalized fluorescence spectra (I i (λ)) have at least a minimum predefined intensity at mutual crossover wavelengths (λ i/i+1 ), wherein the spectrally corrected and normalized fluorescence spectra (I i (λ)) is sequentially arranged;
(b) calculating combination factors (α i ) through statistical averaging of consecutive partial correction functions (F i (λ)), wherein the partial correction functions (F i (λ)) are formed by forming the quotient of the measured fluorescence spectra (J i (λ)) and the corresponding spectrally corrected and normalized fluorescence spectra (I i (λ)) of the fluorescence standards (i), and wherein the statistical averaging takes place over a predefined, limited overlap region (λ i/i+1 ±Δλ OL ) about the mutual crossover wavelengths (λ i/i+1 ), wherein the spectral overlap region (λ i/i+1 ±Δλ OL ; OL=overlap), where the combination factors (α i ) are calculated, corresponds to a range of at most ±12 nm about the mutual crossover wavelengths (λ i/i+1 ); and
(c) determining a total correction function (F(λ)), for the predefined spectral range by combination of the partial correction functions (F i (λ)), which are weighted according to the combination factors (α i ).
14. A method for calibrating a photoluminescence measurement system, in particular a fluorescence measurement system, the method comprising:
(a) measuring fluorescence spectra (J i (λ)) of a plurality of fluorescence standards (i) with the photoluminescence measurement system to be calibrated, wherein the fluorescence standards (i) are selected, so that their combined spectrally corrected and normalized fluorescence spectra (I i (λ)) cover a predefined spectral range in such a way, that the fluorescence bands of the spectrally corrected and normalized fluorescence spectra (I i (λ)) have at least a minimum predefined intensity at mutual crossover wavelengths (λ i/i+1 ), wherein the spectrally corrected and normalized fluorescence spectra (I i (λ)) is sequentially arranged;
(b) calculating combination factors (α i ) through statistical averaging of consecutive partial correction functions (F i (λ)), wherein the partial correction functions (F i (λ)) are formed by forming the quotient of the measured fluorescence spectra (J i (λ)) and the corresponding spectrally corrected and normalized fluorescence spectra (I i (λ)) of the fluorescence standards (i), and wherein the statistical averaging takes place over a predefined, limited overlap region (λ i/i+1 ±Δλ OL ) about the mutual crossover wavelengths (λ i/i+1 ); and
(c) determining a total correction function (F(λ)), for the predefined spectral range by combination of the partial correction functions (F i (λ)), which are weighted according to the combination factors (α i ), wherein the functional dependence of the total correction function (F(λ)) is calculated in a predefined, limited combination region (λ i/i+1 ±Δλ LK ; LK=linkage) about the mutual crossover wavelengths (λ i/i+1 ) through statistical averaging of the weighted, consecutive partial correction functions (F i (λ)).