# Inner Filter Effect Correction for Fluorescence Measurements in Microplates Using Variable Vertical Axis FocusClick to copy article linkArticle link copied!

- Tin Weitner
*****Tin WeitnerFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, Zagreb 10000, Croatia*****Email: [email protected]More by Tin Weitner - Tomislav FriganovićTomislav FriganovićFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, Zagreb 10000, CroatiaMore by Tomislav Friganović
- Davor ŠakićDavor ŠakićFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, Zagreb 10000, CroatiaMore by Davor Šakić

## Abstract

The inner filter effect (IFE) hinders fluorescence measurements, limiting linear dependence of fluorescence signals to low sample concentrations. Modern microplate readers allow movement of the optical element in the vertical axis, changing the relative position of the focus and thus the sample geometry. The proposed *Z*-position IFE correction method requires only two fluorescence measurements at different known vertical axis positions (*z*-positions) of the optical element for the same sample. Samples of quinine sulfate, both pure and in mixtures with potassium dichromate, showed a linear dependence of corrected fluorescence on fluorophore concentration (*R*^{2} > 0.999), up to *A*_{ex} ≈ 2 and *A*_{em} ≈ 0.5. The correction extended linear fluorescence response over ≈98% of the concentration range with ≈1% deviation of the calibration slope, effectively eliminating the need for sample dilution or separate absorbance measurements to account for IFE. The companion numerical IFE correction method further eliminates the need for any geometric parameters with similar results. Both methods are available online at https://ninfe.science.

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### License Summary*

You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:

Creative Commons (CC): This is a Creative Commons license.

Attribution (BY): Credit must be given to the creator.

*Disclaimer

This summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials.

### License Summary*

You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:

Creative Commons (CC): This is a Creative Commons license.

Attribution (BY): Credit must be given to the creator.

*Disclaimer

This summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials.

## Introduction

### Inner Filter Effect in Fluorescence Spectroscopy

*x*–

*y*scanning stage to position each well in the observation path. (2)

### Conventional Methods for IFE Correction

*A*= 0.06, the relative error in recorded fluorescence intensity is approximately 8%, and this difference increases further to 12% at

*A*= 0.1 and 38% at

*A*= 0.3. (5,9) As previously noted by Wang, sample dilution may introduce additional errors and/or alter the chemical properties of the samples. (8)

*F*

_{A}is the absorbance IFE-corrected fluorescence intensity,

*F*

_{1}is the measured (uncorrected) fluorescence intensity,

*A*

_{ex}is the absorbance at the fluorescence excitation wavelength, and

*A*

_{em}is the absorbance at the selected fluorescence emission wavelength. (2)

_{ex}and λ

_{em}must be measured independently. For a detailed overview of the properties of this correction method, the article by Panigrahi and Mishra can be referred. (4) Briefly, the authors described a geometry-dependent maximum of the achievable fluorescence intensity corresponding to a maximum concentration of the analyte, beyond which the observed fluorescence intensity decreases and the emission curve exhibits a downward curvature. They have also shown that the Lakowicz model for the IFE correction is valid only up to

*A*= 0.7. For larger values of

*A*, this model overestimates the loss of observed fluorescence due to IFE, resulting in an upward curvature of the corrected fluorescence. Notwithstanding its limitations, the Lakowicz model is currently extensively used for correcting IFE-related artifacts in the observed fluorescence intensity. (4,8) Therefore, this method was chosen as the benchmark for IFE correction.

*F*

_{0}is the corrected fluorescence intensity and

*F*

_{1}and

*F*

_{2}are the measured fluorescence values for different light path lengths,

*l*

_{1}and

*l*

_{2}. When using the cell shift method proposed by Lutz and Luisi, the values of

*l*

_{1}and

*l*

_{2}are measured along the diagonal in a standard 1 cm rectangular cell. (10) However, this method has limited applicability because it requires special instrumentation that is not commonly available, as noted in the literature. (8,11)

### IFE Correction in Microplates

*z*-axis (perpendicular to the sample well), allowing the sample geometry to be easily changed with the primary goal of optimizing measurement sensitivity. This movement changes the effective light path lengths, with the geometric parameter

*p*corresponding to the distance between the focal point of the measurement and the surface of the liquid in the microplate well. The parameter

*p*can be calculated from the known adjustable

*z*-position of the optical element and other fixed geometrical parameters of the microplate reader (Figure 1) using eq 3

*p*is the distance between the focal point of the measurement and the surface of the liquid in the microplate well (corresponding to the parameter

*l*in eq 2),

*d*is the microplate well depth,

*h*is the distance from the bottom of the microplate well to the surface of the liquid,

*t*is the total height of the microplate,

*f*is the distance from the optical element to the focal point of the lens,

*m*is the depth of the lens slot of the optical element, and

*z*is the distance from the lens to the bottom of the microplate well (

*z*-position).

*d*,

*h*, and

*t*are distinctive for different microplate types, whereas the parameter

*h*also depends on the sample volume in the well. The parameters

*f*and

*m*are distinctive for a particular optical system of the microplate reader instrument. A single overall geometric parameter

*k*for a particular sample volume, microplate, and microplate reader type can be calculated using eq 4

*Z*-position inner filter effect (ZINFE) correction using eq 5

*F*

_{Z}is the ZINFE-corrected fluorescence intensity,

*F*

_{1}and

*F*

_{2}are the measured fluorescence values at different

*z*-positions,

*z*

_{1}and

*z*

_{2}, and

*k*is defined in eq 4.

*k*,

*z*

_{1}, and

*z*

_{2}. In addition to calculations from geometry-dependent parameters, this exponential term can also be obtained by least-squares fitting from experimental values of

*F*

_{1}and

*F*

_{2}, thus obtaining the proposed numerical inner filter effect (NINFE) correction using eq 6

*F*

_{N}is the NINFE-corrected fluorescence intensity based on fluorescence measurements at different

*z*-positions (

*F*

_{1}and

*F*

_{2}), and the exponential term

*N*is obtained by brute-force optimization. This allows a wider range of applicable

*z*-positions and also helps to account for possible reflection effects or errors in the estimation of geometric parameters. For such NINFE correction, only two sets of fluorescence data,

*F*

_{1}and

*F*

_{2}, measured at

*z*-positions

*z*

_{1}and

*z*

_{2}are needed. The actual values of

*z*

_{1}and

*z*

_{2}, or indeed any other geometric parameters, are not necessary to obtain the corrected fluorescence,

*F*

_{N}. This correction can also be applied to data generated by the cell shift method mentioned earlier.

### Objective and Limitations

*et al.*used a custom stage for lateral cuvette movement in order to determine the geometric sensitivity factor of the spectrofluorometer. (3) Gu and Kenny also used a custom stage for cell shift experiments with additional numerical optimization of the geometric parameters, also separately for pIFE and sIFE. (13) However, all these methods are only applicable to conventional spectrofluorometers with detection at a 90° angle in rectangular cuvettes. Moreover, all these methods require separate measurements of sample absorbance and some kind of numerical procedure to account for sample geometry.

*F*

_{1}, with the values of

*F*

_{Z},

*F*

_{N}, and

*F*

_{A}obtained using eqs 5, 6, and 1, respectively. For the first set of experiments, fluorescence and absorbance were measured for the same samples in the same UV-transparent microplates to minimize sample handling. However, the microplates suitable for measuring both UV–vis absorbance and fluorescence and thus a very simple application of eq 1 for the IFE correction are considerably more expensive than non-transparent microplates. To estimate the general applicability of the ZINFE method, which does not require absorbance measurements, all measurements were duplicated using another type of non-transparent microplate as a potentially cost-saving solution.

*z*-axis can be achieved in order to obtain at least two measurements with different

*z*-positions. As far as we know, this is the first attempt at IFE correction specifically intended for measurements in microplates.

## Experimental Section

*A*

_{ex}≈ 2, which is acceptable for most spectrophotometers and should be common in most experimental setups. In the experiments with added PD, the concentrations were chosen so that the maximum concentration of PD corresponds to

*A*

_{ex}≈ 1. Full details on reagents and sample preparation can be found in Section 2 of the Supporting Information.

*z*-position. For the

*z*-position IFE corrections (ZINFE, eq 5), the measured fluorescence intensity values (

*F*

_{1}) obtained for each

*z*-position were corrected using the fluorescence intensity values (

*F*

_{2}) obtained for the remaining

*z*-positions. A total of

*n*(

*n*– 1) = 72 corrections were obtained. As a measure of linearity, the

*R*

^{2}statistic was calculated for each data set. The

*z*-position correction whose

*R*

^{2}value was closest to 1 was selected as optimal and used to compare the results. (12,13)

*N*corresponding to the optimal combination of positions

*z*

_{1}and

*z*

_{2}found by the procedure described above was chosen as the starting point (seed) for numerical optimization. This starting point is then varied in a series of 20 steps with a step size of 1 in both the positive and negative directions to produce a series of

*R*

^{2}values. An exponent corresponding to the maximum

*R*

^{2}value is then used as the seeding point in the next optimization cycle with the same number of steps in both directions, while the step size is decreased by a factor of 10. This procedure continues for 10 cycles or when the difference between the exponents from successive cycles is Δ

*N*< 1 × 10

^{–6}, whichever comes first.

*z*-position (

*z*

_{1}) used for the best

*z*-position correction. Therefore, for each concentration series in a given microplate, all values are derived from the same value of

*F*

_{1}(corresponding to the uncorrected data) used in eqs 1, 5, and 6. For data processing, a dedicated script was written in the Javascript programming language. (15) Full details on background correction and other data processing, including statistical considerations, can be found in the Supporting Information, Section 3.

sample^{a} | plate type^{b} | correction type^{c} | R^{2} | b %^{d} | LOD %^{e} | z_{1}/mm | Δz^{f}/mm | c_{max}^{g}/μM | A_{max}^{h} (λ_{ex}, λ_{em}) |
---|---|---|---|---|---|---|---|---|---|

Q | T (data set 1) | F_{1} | 0.87449 | 17.5 | 36.4 | 19.0 | 2.0 | 679.3 | 1.984, 0.158 |

F_{Z} | 0.99980 | 0.54 | 1.39 | ||||||

F_{N} | 0.99984 | 0.24 | 1.20 | ||||||

F_{A} | 0.95074 | –7.87 | 21.9 | ||||||

NT (data set 2) | F_{1} | 0.81861 | 21.7 | 45.2 | 18.0 | 2.5 | |||

F_{Z} | 0.99971 | 0.12 | 1.64 | ||||||

F_{N} | 0.99973 | –0.08 | 1.59 | ||||||

Q-v | T (data set 3) | F_{1} | 0.81967 | 21.3 | 45.1 | 19.0 | 2.0 | 316.0 | 1.873, 0.443 |

F_{Z} | 0.99951 | 0.95 | 2.13 | ||||||

F_{N} | 0.99964 | 0.43 | 1.83 | ||||||

F_{A} | 0.93753 | –8.15 | 24.8 | ||||||

NT (data set 4) | F_{1} | 0.73752 | 25.9 | 57.3 | 18.0 | 2.0 | |||

F_{Z} | 0.99974 | 0.47 | 1.55 | ||||||

F_{N} | 0.99979 | 0.14 | 1.38 | ||||||

Q-f | T (data set 5) | F_{1} | 0.98744 | 5.39 | 10.8 | 18.0 | 1.0 | 312.9 | 1.921, 0.464 |

F_{Z} | 0.99959 | –0.12 | 1.94 | ||||||

F_{N} | 0.99965 | 0.22 | 1.80 | ||||||

F_{A} | 0.98111 | –4.85 | 13.3 | ||||||

NT (data set 6) | F_{1} | 0.98918 | 4.93 | 10.0 | 18.0 | 3.0 | |||

F_{Z} | 0.99964 | 1.24 | 1.83 | ||||||

F_{N} | 0.99972 | 0.89 | 1.61 |

^{a}

Q corresponds to the pure QS concentration series; Q-v corresponds to the variable concentration of the absorber PD; Q-f corresponds to the fixed total concentration of PD.

^{b}

T corresponds to the UV-transparent microplates; NT corresponds to the non-transparent microplates. Data set numbers correspond to the averaged triplicate data preformatted for automated processing. (16)

^{c}

*F*_{1} corresponds to uncorrected fluorescence; *F*_{Z} corresponds to ZINFE-corrected fluorescence intensity (eq 5); *F*_{A} corresponds to absorbance IFE-corrected fluorescence intensity (eq 1); *F*_{N} corresponds to NINFE-corrected fluorescence intensity.

^{d}

Percent error of the normalized data slope with respect to the IFS. The values of slope and intercept used for data normalization for each concentration series are given in Table S12, Supporting Information.

^{e}

LOD (α = β = 0.05); the values were normalized as percentage of *c*_{max}.

^{f}

Defined as Δ*z* = *z*_{2} – *z*_{1}, where *z*_{1} and *z*_{2} are the different *z*-positions used for measurements of *F*_{1} and *F*_{2} (eq 5).

^{g}

Maximum concentration of QS in the concentration series.

^{h}

Maximum absorbance at the excitation and emission wavelengths, *λ*_{ex} = 345 nm and *λ*_{em} = 390 nm, respectively.

## Results and Discussion

*F*

_{1}and

*F*

_{2}deviate from linearity due to IFE caused by increasing sample concentration. Although both

*F*

_{1}and

*F*

_{2}are recorded for the same samples in the same microplate, they are measured at different

*z*-positions, resulting in different sample geometries and different dependences of the measured fluorescence on sample concentration. However, the values of

*F*

_{1}and

*F*

_{2}obtained in this way can be used to calculate the corrected

*F*with improved linearity according to eqs 5 or 6. The corresponding results for all concentration series can be found in Figure S9, Supporting Information.

*F*

_{x,norm}=

*A*

_{ex}/

*A*

_{max}, where

*A*

_{ex}is the baseline-corrected absorbance at the excitation wavelength and

*A*

_{max}is the maximum value of

*A*

_{ex}for the given concentration range; (ii) ordinate values were calculated as

*c*

_{norm}=

*F*

_{x}/(

*a*×

*c*

_{max}+

*b*), where

*F*

_{x}corresponds to either the uncorrected or corrected fluorescence (

*F*

_{1},

*F*

_{Z},

*F*

_{N}or

*F*

_{A}) and

*a*and

*b*are the slope and intercept, respectively, of the linear regression line for the corresponding data (Table S12, Supporting Information). The normalized values are 0 <

*c*

_{norm}< 1 and 0 <

*F*

_{x,norm}< ≈ 1, with maximum

*F*

_{x,norm}values depending on the deviation of the normalized value of

*F*

_{A},

*F*

_{Z}, or

*F*

_{N}compared with the slope of the ideal fluorescence signal (IFS).

*F*and

*A*in the absence of IFE. (17,18) The slope of this linear relationship depends on the structural characteristics of the fluorophore, and the intercept should be equal to 0 after accounting for background fluorescence and absorbance

*via*blank subtraction. (19) Therefore, the value of IFS for the normalized data (i.e., plots of

*F*

_{x,norm}vs

*c*

_{norm}) is a line with slope

*a*= 1 and intercept

*b*= 0, which allows very easy comparison of the uncorrected or corrected data with the ideal measurement response. A better match of the normalized data with the IFS requires a smaller deviation of the slope and the intercept of the linear regression from the values

*a*= 1 and

*b*= 0, respectively.

*a*+

*b*= 1 is valid for all normalized data, the value of

*b*was given as a suitable measure of linearity and accuracy for comparing the different correction methods (Table 1). The values of

*b*can be either positive or negative, corresponding to the downward or upward curvature of the fluorescence signal, respectively. In addition, the value of

*b*obtained by the described normalization is numerically equal but opposite in sign to the percent error of the slope of the line of corrected fluorescence (

*mErr*%, eq S7, Supporting Information), which was used by Gu and Kenny to compare IFE corrections. (13) Therefore, the values of

*b*were also expressed as

*b*%, which means the percent error of the slope of the normalized data from IFS.

*s*

_{y}, defined in eq S2, Supporting Information). For convenient comparison of the results, the LOD values obtained for the raw data (Table S12, Supporting Information) were normalized as a percentage of the highest concentration of the analyte in the corresponding series (

*c*

_{max}), resulting in LOD % values (Table 1).

### Uncorrected Fluorescence (*F*_{1})

*F*

_{1}) for the QS concentration series show a clear deviation from linearity, except for the Q-f concentration series, that is, for a fixed total concentration of PD (Figure S8, Supporting Information). The linearity of the uncorrected data depends on the

*z*-position at which the fluorescence intensity was measured for all QS concentration series and increases with

*z*-position (Figure S14, Supporting Information). The best

*R*

^{2}values are observed at

*z*= 21 mm for all concentration series, which is consistent with increasing linearity of the fluorescence signal as the light path length decreases (higher

*z*-values correspond to shorter light path lengths), that is, lower effective absorbance and thus smaller IFE. All deviations from the ideal signal were positive (i.e.,

*b*> 0, Figure 3, right), corresponding to a downward curvature for all concentration series due to IFE.

*z*-position corresponding to the

*z*

_{1}value for the best ZINFE correction yield values of

*R*

^{2}< 0.875 and large values of LOD % > 36% of

*c*

_{max}and

*b*% > 17%, consistent with the observed downward curvature of the fluorescence signal (Table 1, Q and Q-v concentration series). The Q-f concentration series gave slightly better results (

*R*

^{2}> 0.98, LOD % ≈ 10% of

*c*

_{max}and

*b*% ≈ 5%), consistent with the observed lower curvature of the fluorescence signal compared with the Q and Q-v concentration series. The Q-f concentration series also showed the lowest dependence of

*R*

^{2}values on

*z*-position (0.971 <

*R*

^{2}< 0.995, Figure S14, Supporting Information). Similar results for uncorrected data were also obtained for non-transparent microplates (all results can be found in Tables 1 and S12, Supporting Information). This observation can most likely be attributed to lower variability in total absorbance at the excitation wavelength for this concentration series compared to others (Figure S11 and Table S22, Supporting Information).

### Absorbance IFE-corrected Fluorescence (*F*_{A})

*z*-positions were obtained for UV-transparent microplates only (measured absorbance data can be seen in Figure S11, Supporting Information). Each absorbance IFE correction (

*F*

_{A}, eq 1) gave better linearity than the uncorrected data, except for the Q-f concentration series (Table 1).

*z*-position at which fluorescence intensity was measured for all concentration series and decreases with

*z*-position (Figure S15, Supporting Information). The variation in

*R*

^{2}values is smaller and also inverse to the dependence observed for uncorrected data (Figure S14, Supporting Information). This observation is consistent with increasing linearity of the absorbance-corrected fluorescence signal with increasing light path length (lower

*z*-values correspond to longer light path lengths); that is, the effective absorbance approaches the value used for the correction. Notably, the correction factor in eq 2, (

*A*

_{ex}+

*A*

_{em})/2, is independent of

*z*-position.

*R*

^{2}≈ 0.99 and LOD % ≈ 10%, giving a linear response over approximately 90% of the concentration range with less than 3.5% deviation of the calibration slope from the ideal signal. All deviations from the ideal signal were negative (i.e.,

*b*< 0, Figure 3, right), corresponding to an upward curvature for all concentration series due to overcorrection (i.e., overestimated fluorescence loss) associated with the Lakowicz model, especially at higher absorbance. (4) The Q-f concentration series again showed the least dependence of

*R*

^{2}values on

*z*-position (0.975 <

*R*

^{2}< 0.989, Figure S15, Supporting Information). The absorbance IFE correction decreases the LOD % values by approximately 40%, compared to the uncorrected data for the Q and Q-v concentration series. Surprisingly, the LOD % value was increased by 20% compared to the uncorrected data for the Q-f concentration series, indicating that this type of correction is not appropriate in the presence of a background absorber.

### ZINFE-corrected Fluorescence (*F*_{Z})

*z*-positions. The optimal

*z*-position IFE correction (

*F*

_{Z}, eq 5) significantly improves the linearity of the fluorescence signal for all QS concentration series, yielding values of

*R*

^{2}> 0.999 and deviation from the ideal signal response in the range of −0.122 <

*b*% < 1.243. The LOD % values for all concentration series were in the range of 1.358–2.130% of the

*c*

_{max}. Therefore, a linear response was obtained for all concentration series over approximately 98% of the concentration range with a maximum deviation of the calibration slope from the ideal signal of approximately 1% (Figure 3). For comparison, the uncorrected data at the same

*z*-position for the entire concentration range gave values of

*R*

^{2}< 0.9, except for the Q-f series, which gave values of

*R*

^{2}< 0.99. The deviations from the ideal signal were much worse for the uncorrected data (

*b*% ≈ 20% for the Q and Q-v concentration series, and

*b*% ≈ 5% for the Q-f concentration series) and also for the absorbance-corrected values (

*b*% ≈ −5%).

*z*-position correction depends largely on the choice of

*F*

_{1}and

*F*

_{2}(i.e., the measured fluorescence values at different

*z*-positions) used in eq 5. However, each ZINFE correction gave better linearity than the uncorrected data, and the best overall

*R*

^{2}value is obtained with the

*z*-position correction. The three-dimensional plots for the dependence of the linear regression model error, calculated as Δ

*R*= −1/(1 –

*R*

^{2}), on the values of

*z*

_{1}and

*z*

_{2}showed a complex surface with multiple minima for all concentration series (Figure S13, Supporting Information). Such a shape of the error surface seems to justify the attempt of further numerical optimization according to eq 6.

### NINFE-corrected Fluorescence (*F*_{N})

*k*,

*z*

_{1}, and

*z*

_{2}, which yielded the highest

*R*

^{2}value, show a slight improvement compared with the calculation using geometry-dependent parameters (Table S16, Supporting Information). The exponents obtained from the geometric parameters and numerical optimization are in good agreement for Q and Q-v concentration series, with relatively small differences between the exponents (approximately 0.05), whereas slightly larger differences were obtained for the Q-f concentration series (approximately 0.2) (Table S16, Supporting Information). In general, the exponent optimization curves (Figure S17, Supporting Information) show remarkable similarity between the calculated and numerically optimized exponent values.

*R*

^{2}values were increased in the fourth or fifth decimal range, while the LOD % and

*b*% were improved by approximately 0.5%, except for a single data set (

*b*% was larger for Q-f concentration series in UV-transparent microplates).

### Transparent Versus Non-transparent Microplates and the Effect of Background Correction

*b*% values for all

*F*

_{Z}corrections were comparable for all concentration series, with slightly better values obtained by numerical optimization (

*F*

_{N}). A particularly interesting feature of the ZINFE correction or the NINFE correction is the ability to use fluorescence data without background correction. The results obtained for such data gave only slightly worse results, again with values of

*R*

^{2}> 0.999 for all concentration series with approximately 0.5% higher values of LOD and 0.4% higher absolute values of

*b*%, compared with the data with background correction (Table S18 and Figures S19 and S20, Supporting Information). However, data without background correction should be used with caution because different behaviors of the background signal can be expected for samples other than those described here.

### IFE Correction for Low-Concentration Samples

*F*

_{1}) is very linear (

*R*

^{2}> 0.994) for the first seven points in each concentration series. However, even for this concentration range, slightly increased

*R*

^{2}values and lower

*b*% values were observed for the ZINFE- and NINFE-corrected data for the Q and Q-v concentration series in both UV-transparent and non-transparent microplates (Table S21, Supporting Information). Slightly decreased

*R*

^{2}values and higher

*b*% values were observed for the Q-f concentration series for all IFE corrections, which may be attributed to increased noise due to the use of two measured values instead of only one. This is an indication that the regression residuals at low sample concentrations are mainly due to measurement errors rather than IFE.

## Conclusions

*A*

_{ex}≈ 2 and

*A*

_{em}≈ 0.5, with possible applicability at higher absorbance values. A simple heuristic for performing the measurements is to select a set of available

*z*-positions depending on the characteristics of the microplate reader and find the optimal combination of

*z*

_{1}and

*z*

_{2}based on the quality of the linearization. In general, for this particular experimental setup, the best combinations of

*z*-positions yielding the highest

*R*

^{2}values were obtained with

*F*

_{1}values measured at

*z*

_{1}= 18 or

*z*

_{1}= 19, while the

*F*

_{2}values are measured at 1–3 mm lower values of

*z*

_{2}(lower

*z*-values correspond to a longer light path length).

*z*-positions for the measurements. Moreover, NINFE can be used not only for measurements in microplate readers but also for any measurements obtained by the cell shift method. However, this method can be considered as a black-box system that may not be suitable for all users, who may then prefer to use the ZINFE method with known geometric parameters.

*b*%| < 1.3% for all concentration series, Figure 4). In addition, we have shown that both ZINFE and NINFE are comparably effective for samples with an additional absorber in varying proportions. Similarly, we have shown that both methods are comparably effective in both UV-transparent and non-transparent microplates. The extended linear response of the fluorescence signal provided by ZINFE or NINFE allows simplified fluorescence measurements without sample dilution, thus eliminating the often complex and time- and resource-consuming liquid handling associated with microplates.

## Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.2c01031.

Additional experimental details including instrumental parameters, sample preparation, statistical considerations, and results for all data sets (PDF)

## Terms & Conditions

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## Acknowledgments

This work was supported by funding from the Croatian Science Foundation grant UIP-2017-05-9537─Glycosylation as a factor in the iron transport mechanism of human serum transferrin (GlyMech). Additional support was provided by the European Structural and Investment Funds for the “Croatian National Centre of Research Excellence in Personalized Healthcare” (contract #KK.01.1.1.01.0010), “Centre of Competences in Molecular Diagnostics” (contract #KK.01.2.2.03.0006), and the European Regional Development Fund grant for “Strengthening of Scientific Research and Innovation Capacities of the Faculty of Pharmacy and Biochemistry at the University of Zagreb” (FarmInova, contract #KK.01.1.1.02.0021).

## References

This article references 21 other publications.

**1**Valeur, B.; Berberan-Santos, M. N.*Molecular Fluorescence: Principles and Applications*, 2nd; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2013.Google ScholarThere is no corresponding record for this reference.**2**Lakowicz, J. R. Instrumentation for Fluorescence Spectroscopy.*Principles of Fluorescence Spectroscopy*; Springer US: Boston, MA, 2006; pp 27– 61.Google ScholarThere is no corresponding record for this reference.**3**Kimball, J.; Chavez, J.; Ceresa, L.; Kitchner, E.; Nurekeyev, Z.; Doan, H.; Szabelski, M.; Borejdo, J.; Gryczynski, I.; Gryczynski, Z. On the Origin and Correction for Inner Filter Effects in Fluorescence Part I: Primary Inner Filter Effect-the Proper Approach for Sample Absorbance Correction.*Methods Appl. Fluoresc.*2020,*8*, 033002, DOI: 10.1088/2050-6120/ab947cGoogle Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1ajsb3J&md5=9c8f9e5cea63b21d8923ee951d011bbfOn the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correctionKimball, Joseph; Chavez, Jose; Ceresa, Luca; Kitchner, Emma; Nurekeyev, Zhangatay; Doan, Hung; Szabelski, Mariusz; Borejdo, Julian; Gryczynski, Ignacy; Gryczynski, ZygmuntMethods and Applications in Fluorescence (2020), 8 (3), 033002CODEN: MAFEB2; ISSN:2050-6120. (IOP Publishing Ltd.)Fluorescence technologies have been the preferred method for detection, anal. sensing, medical diagnostics, biotechnol., imaging, and gene expression for many years. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biol. samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. However, many previous approaches have given only approx. exptl. methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of obsd. results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide. The procedure is generic and applies to many other applications like quantum yield detn., tissue/blood absorption, or acceptor absorption in FRET expts.**4**Panigrahi, S. K.; Mishra, A. K. Study on the Dependence of Fluorescence Intensity on Optical Density of Solutions: The Use of Fluorescence Observation Field for Inner Filter Effect Corrections.*Photochem. Photobiol. Sci.*2019,*18*, 583– 591, DOI: 10.1039/C8PP00498FGoogle Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktFymsA%253D%253D&md5=f636a8cc0938a2740da1cedef7973887Study on the dependence of fluorescence intensity on optical density of solutions: the use of fluorescence observation field for inner filter effect correctionsPanigrahi, Suraj Kumar; Mishra, Ashok KumarPhotochemical & Photobiological Sciences (2019), 18 (2), 583-591CODEN: PPSHCB; ISSN:1474-905X. (Royal Society of Chemistry)In this study, we report the identification of absorbance value of an analyte at the excitation wavelength that corresponds to the max. of the obsd. fluorescence intensity obtainable for a certain instrument operating with right-angle fluorescence measurement ([Formula Omitted]). The [Formula Omitted] value depends on the fluorescence observation field (FOF) dimensions of the concerned spectrofluorometer. As the FOF varies from instrument to instrument, this study presents a simple method for obtaining FOF dimensions. Using the knowledge of FOF, absorbance of analyte at the excitation wavelength (Aλex) and emission wavelength (Aλem), we deduced a derived absorbance spectral parameter (Dabs). The obsd. fluorescence intensity of an analyte is proportional to the Dabs. While differentiating Dabs w.r.t. Aλex, the value of [Formula Omitted] for the concentred spectrofluorometer was obtained and subsequently could be used for maximizing fluorescence sensitivity. It was obsd. that when the FOF was a point at the center of a 1 cm path-length cuvette, the ♂[Formula Omitted] value was 0.87 with a progressive widening of FOF, the ♂[Formula Omitted] value increased gradually till ∼1.0. The proposed methodol. was established using two well-known inner filter effect (IFE) correction models (Parker and Lakowicz model). The Dabs obtained from the Parker model corresponded well with the obsd. fluorescence data; however, the Dabs obtained using the Lakowicz model overestimated the loss of fluorescence because of IFE. Using equations derived from the Parker model, the correction of obsd. fluorescence intensity for IFE could be achieved. Furthermore, it is demonstrated that the commonly used the Lakowicz model loses its correction efficiency at absorbance values of ≥0.7.**5**Fonin, A. V.; Sulatskaya, A. I.; Kuznetsova, I. M.; Turoverov, K. K. Fluorescence of Dyes in Solutions with High Absorbance. Inner Filter Effect Correction.*PLoS One*2014,*9*, e103878 DOI: 10.1371/journal.pone.0103878Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1eqt7zN&md5=1465d55a4ac805934a9bf9253e2bb8cfFluorescence of dyes in solutions with high absorbance. Inner filter effect correctionFonin, Alexander V.; Sulatskaya, Anna I.; Kuznetsova, Irina M.; Turoverov, Konstantin K.PLoS One (2014), 9 (7), e103878/1-e103878/8, 8 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Fluorescence is a proven tool in all fields of knowledge, including biol. and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concn. that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dil. solns. We showed that correction must be performed always. Furthermore, high-concn. solns. (high absorbance) are inherent condition in studying of the photophys. properties of fluorescent dyes and the functionally significant interactions of biol. macromols. We proposed an easy to use method to correct the exptl. recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye mols. do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of exptl. detected total fluorescence intensity of the dye on its absorbance coincides with the calcd. dependence and the correction factor for eliminating the primary inner filter effect can be calcd. on the basis of soln. absorbance. It was exptl. shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and addnl. spectral anal. allow to conclude that the most probable reason of the deviation of exptl. detected fluorescence intensity dependence on soln. absorbance from the calcd. dependence is the dye mols. self-quenching, which accompanies resonance radiationless excitation energy transfer.**6**Ma, C.; Li, L.; Gu, J.; Zhu, C.; Chen, G. Inflection Point of the Fluorescence Excitation Spectra Induced by Secondary Inner Filter Effect.*Spectrosc. Lett.*2018,*51*, 319– 323, DOI: 10.1080/00387010.2018.1461655Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1ygurbF&md5=eba2a4f0d8ff4ea325a173fbfda4564bInflection point of the fluorescence excitation spectra induced by secondary inner filter effectMa, Chaoqun; Li, Lei; Gu, Jiao; Zhu, Chun; Chen, GuoqingSpectroscopy Letters (2018), 51 (7), 319-323CODEN: SPLEBX; ISSN:0038-7010. (Taylor & Francis, Inc.)The secondary inner filter effect on the fluorescence excitation spectra of rhodamine 6G aq. solns. with concn. was demonstrated in this paper. The peak of fluorescence excitation spectrum stays at 525 nm at low concns., while it breaks up and turns into valley at high concns. The threshold concn. was detd. to be 3.16 mg/L by the second deriv. spectroscopy. A math. model was proposed to explain the inflection point of fluorescence excitation spectrum caused by the secondary inner filter effect. On the basis of it, the threshold concn. was calcd. to be 2.86 mg/L, approaching to the exptl. result.**7**Panigrahi, S. K.; Mishra, A. K. Inner Filter Effect in Fluorescence Spectroscopy: As a Problem and as a Solution.*J. Photochem. Photobiol., C*2019,*41*, 100318, DOI: 10.1016/j.jphotochemrev.2019.100318Google ScholarThere is no corresponding record for this reference.**8**Wang, T.; Zeng, L.-H.; Li, D.-L. A Review on the Methods for Correcting the Fluorescence Inner-Filter Effect of Fluorescence Spectrum.*Appl. Spectrosc. Rev.*2017,*52*, 883– 908, DOI: 10.1080/05704928.2017.1345758Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1OmtrbM&md5=9908fcde03ff0b1bf840393460767b5fA review on the methods for correcting the fluorescence inner-filter effect of fluorescence spectrumWang, Tan; Zeng, Li-Hua; Li, Dao-LiangApplied Spectroscopy Reviews (2017), 52 (10), 883-908CODEN: APSRBB; ISSN:0570-4928. (Taylor & Francis, Inc.)Fluorescence spectroscopy is frequently used to analyze the concn. of fluorescent materials in soln. However, in conventional fluorescence spectroscopy, the response between the fluorescence intensity and fluorophore concn. is nonlinear at high concns. due to uncompensated inner-filter effects (IFE). Many methods to resolve this problem have been developed in recent decades. This review introduces the methods used to correct the IFE, including direct correction and parameter correction. Relevant detection parameters, including the materials, matrixes, detection limits, detection instruments and relative std. deviations, are tabulated. The advantages and limitations of these correction techniques are also discussed. Finally, the methods used to correct for the IFE are summarized, and future research directions are discussed.**9**Kubista, M.; Sjöback, R.; Eriksson, S.; Albinsson, B. Experimental Correction for the Inner-Filter Effect in Fluorescence Spectra.*Analyst*1994,*119*, 417– 419, DOI: 10.1039/AN9941900417Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXivV2ju7w%253D&md5=8eb5dd1c01b9239ef96a3a2700d5cb60Experimental correction for the inner-filter effect in fluorescence spectraKubista, Mikael; Sjoback, Robert; Eriksson, Svante; Albinsson, BoAnalyst (Cambridge, United Kingdom) (1994), 119 (3), 417-19CODEN: ANALAO; ISSN:0003-2654.Recorded fluorescence intensity is in general not proportional to sample concn. owing to absorption of the incident and emitted light passing through the sample to and from the point inside the cell where the emission is detected. This well known inner-filter effect depends on sample absorption and on instrument geometry, and is usually significant even in samples with rather low absorption (the error is about 8% at an absorbance of 0.06 in a 1 cm square cell). In this work the authors show that a particular exptl. set-up can be calibrated for the inner-filter effect from the absorption and fluorescence excitation spectra of a suitable std. The calibration takes only a few minutes and provides correction with sufficient accuracy for most practical situations.**10**Lutz, H.-P.; Luisi, P. L. Correction for Inner Filter Effects in Fluorescence Spectroscopy.*Helv. Chim. Acta*1983,*66*, 1929– 1935, DOI: 10.1002/hlca.19830660704Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXmt1Wls78%253D&md5=a37690e4d357b4bd6f1e85e40c77edfeCorrection for inner filter effects in fluorescence spectroscopyLutz, Hans Peter; Luisi, Pier LuigiHelvetica Chimica Acta (1983), 66 (7), 1929-35CODEN: HCACAV; ISSN:0018-019X.A method is proposed for correcting exptl. fluorescence readings for inner filter effects, i.e., the absorption of the exciting light and/or the absorption of the emitted radiation, which cause the nonlinearity between fluorescence intensity and fluorophor concn. Basically the method consists of measuring the fluorescence intensity at 2 different points along the diagonal in the cell. Unlike similar methods proposed in the literature, the 2 points are cor. simultaneously for both absorption of excitation and of emission radiation without the necessity of reading the optical d. of the soln., and with a very simple data elaboration.**11**Puchalski, M. M.; Morra, M. J.; von Wandruszka, R. Assessment of Inner Filter Effect Corrections in Fluorimetry.*Fresenius. J. Anal. Chem.*1991,*340*, 341– 344, DOI: 10.1007/BF00321578Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXlsV2ns7s%253D&md5=6bcb13045993d63a5b79e46c78d417e4Assessment of inner filter effect corrections in fluorimetryPuchalski, M. M.; Morra, M. J.; Von Wandruszka, R.Fresenius' Journal of Analytical Chemistry (1991), 340 (6), 341-4CODEN: FJACES; ISSN:0937-0633.The inner filter effect (IFE) in fluorescence spectroscopy is not easily distinguished from dynamic and static quenching phenomena, since IFE rarely occurs without quenching. IFE corrections may be subject to under- or over-compensation effects that are difficult to assess accurately. To evaluate existing IFE correction procedures, it is proposed that the linearity of resulting Stern-Volmer plots and the relative change of their slopes with temp. be adopted as criteria. Three correction methods are assayed in this manner, and the equation described by T. Gauthier et al. (1986) is found to produce the best results.**12**Kasparek, A.; Smyk, B. A New Approach to the Old Problem: Inner Filter Effect Type I and II in Fluorescence.*Spectrochim. Acta, Part A*2018,*198*, 297– 303, DOI: 10.1016/j.saa.2018.03.027Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXlt1Sgs7k%253D&md5=cad0341182ea4ab2108c638e2d23ec04A new approach to the old problem: Inner filter effect type I and II in fluorescenceKasparek, Adam; Smyk, BogdanSpectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy (2018), 198 (), 297-303CODEN: SAMCAS; ISSN:1386-1425. (Elsevier B.V.)The fluorescence technique is very popular and has been used in many fields of research. It is simple in its assumptions but not very easy to use. One of the main problems is the inner filter effect (IF) I and II which takes place in the cuvette. IF type I is permanently present, but IF type II occurs only when absorption and fluorescence spectra overlap. To avoid IF type I, absorbencies in the cuvette should be smaller than 0.05, which is however very difficult to obtain in many expts. The authors propose a new method to solve these problems in the case of a Cary Eclipse fluorometer, having horizontally-oriented slits, based on old equations developed in the middle of the last century. This method can be applied for other instruments, even these with vertically-oriented beams, because the authors share scripts written in MATLAB and GRAMS/AI environment. Calcns. in the authors' method enable specifying beam geometry parameters in the cuvette, which is necessary to obtain the correct shape and fluorescence intensity of emission and excitation spectra. Such a specific fluorescence intensity dependence on absorbance can, in many cases, afford possibilities to det. the quantum yield (QY) using slopes of the straight-lines, which was demonstrated using Tryptophan (Trp), Tyrosine (Tyr), and Rhodamine B (RhB) solns. For example, assuming that QY = 0.14 for Tyr, the QY detd. for RhB reached QY = 0.71 ± 0.05, although the measurement for Tyr and RhB was performed at a completely different spectral range.**13**Gu, Q.; Kenny, J. E. Improvement of Inner Filter Effect Correction Based on Determination of Effective Geometric Parameters Using a Conventional Fluorimeter.*Anal. Chem.*2009,*81*, 420– 426, DOI: 10.1021/ac801676jGoogle Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVOnsLrN&md5=08dff6ebf7830a79bb2eea9afc7e58a3Improvement of Inner Filter Effect Correction Based on Determination of Effective Geometric Parameters Using a Conventional FluorimeterGu, Qun; Kenny, Jonathan E.Analytical Chemistry (Washington, DC, United States) (2009), 81 (1), 420-426CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The most widely used correction of fluorescence intensities for inner filter effects in conventional (90°) fluorimeters fails at high absorbance values. We have critically examd. this failure, which is caused by the difference between the geometrical parameters (GPs) of the excitation and emission beams in the typical instrument (focused beams) and in the theor. picture on which the correction is based (collimated beams). We provide two types of exptl. measurement of GPs and show that their substitution in the correction equations leads to significant improvements in the linear range of cor. fluorescence. We also demonstrate that math. optimizations give greater improvements and that the optimizations yield GPs consistent with exptl. measurements. For solns. exhibiting primary inner filter effect only, we have extended the range of linearity of cor. fluorescence to aex (absorbance per cm) up to 5.3; for systems with both primary and secondary inner filter effects we have achieved linearity for aex + aem = 6.7. In all cases linear fits have slopes which agree well with the dil. limit. Different series of one- and two-solute solns. were used to demonstrate effectiveness of our correction methods. We also provide a rationale for the unexpected independence of GPs on excitation and emission bandwidths.**14**Tucker, S. A.; Amszi, V. L.; Acree, W. E. Primary and Secondary Inner Filtering. Effect of K2Cr2O7 on Fluorescence Emission Intensities of Quinine Sulfate.*J. Chem. Educ.*1992,*69*, A8, DOI: 10.1021/ed069pA8Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xht1Oktrs%253D&md5=ac3843f19ffd2848d89269aa6eb0123cPrimary and secondary inner filtering: effect of potassium dichromate on fluorescence emission intensities of quinine sulfateTucker, Sheryl A.; Amszi, Vicki L.; Acree, William E., Jr.Journal of Chemical Education (1992), 69 (1), A8, A11-A12CODEN: JCEDA8; ISSN:0021-9584.A lab. expt. is described in which the effect of primary and secondary inner filtering is illustrated by measuring the fluorescence emission signals at 450 nm of 4 ppm quinine sulfate solns. contg. 0, 0.025, 0.05, 0.1, 0.15, and 0.2 mg/mL of K2Cr2O7.**15**Šakić, D.; Weitner, T.; Friganović, T. NINFE. https://ninfe.science/.Google ScholarThere is no corresponding record for this reference.**16**Weitner, T.; Friganović, T.; Šakić, D.*Inner Filter Effect Correction for Fluorescence Measurements in Microplates*, 2022, DOI: 10.5281/zenodo.5849302 .Google ScholarThere is no corresponding record for this reference.**17**Holland, J. F.; Teets, R. E.; Kelly, P. M.; Timnick, A. Correction of Right-Angle Fluorescence Measurements for the Absorption of Excitation Radiation.*Anal. Chem.*1977,*49*, 706– 710, DOI: 10.1021/ac50014a011Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2sXhsl2rsrc%253D&md5=33b521b8ff03ca7dc5fd3a12e164750aCorrection of right-angle fluorescence measurements for the absorption of excitation radiationHolland, John F.; Teets, Richard E.; Kelly, Patrick M.; Timnick, AndrewAnalytical Chemistry (1977), 49 (6), 706-10CODEN: ANCHAM; ISSN:0003-2700.The effect of excitation beam absorption on measured values of fluorescence was studied with a computer-centered spectrofluorimeter capable of measuring fluorescence and absorbance simultaneously. This effect appears to be independent of the nature of the absorbing species and the excitation and emission wavelengths. A model is proposed and tested which corrects fluorescence, obsd. at 90°, for the attenuation of the excitation beam caused by the absorbance of the fluorophore and any chromophores present in the cell. The resulting absorption-cor. fluorescence is linear with the concn. of the fluorophore in solns. with total absorbances as high as 2.0.**18**Miller, J. N. Inner Filter Effects, Sample Cells and Their Geometry in Fluorescence Spectrometry. In*Standards in Fluorescence Spectrometry: Ultraviolet Spectrometry Group*; Miller, J. N., Ed.; Techniques in Visible and Ultraviolet Spectometry; Springer Netherlands: Dordrecht, 1981; pp 27– 43.Google ScholarThere is no corresponding record for this reference.**19**Kothawala, D. N.; Murphy, K. R.; Stedmon, C. A.; Weyhenmeyer, G. A.; Tranvik, L. J. Inner Filter Correction of Dissolved Organic Matter Fluorescence.*Limnol. Oceanogr.: Methods*2013,*11*, 616– 630, DOI: 10.4319/lom.2013.11.616Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWhsr7M&md5=e2707da6f3d995ef3c45b15a43ef0658Inner filter correction of dissolved organic matter fluorescenceKothawala, Dolly N.; Murphy, Kathleen R.; Stedmon, Colin A.; Weyhenmeyer, Gesa A.; Tranvik, Lars J.Limnology and Oceanography: Methods (2013), 11 (Nov.), 616-630, 15 pp.CODEN: LOMIBY; ISSN:1541-5856. (American Society of Limnology and Oceanography)The fluorescence of dissolved org. matter (DOM) is suppressed by a phenomenon of self-quenching known as the inner filter effect (IFE). Despite widespread use of fluorescence to characterize DOM in surface waters, the advantages and constraints of IFE correction are poorly defined. We assessed the effectiveness of a commonly used absorbance-based approach (ABA), and a recently proposed controlled diln. approach (CDA) to correct for IFE. Linearity between cor. fluorescence and total absorbance (ATotal; the sum of absorbance at excitation and emission wavelengths) across the full excitation-emission matrix (EEM) in diln. series of four samples indicated both ABA and CDA were effective to an absorbance of at least 1.5 in a 1 cm cell, regardless of wavelength positioning. In regions of the EEMs where signal to background noise (S/N) was low, CDA correction resulted in more variability than ABA correction. From the ABA algorithm, the onset of significant IFE (>5%) occurs when absorbance exceeds 0.042. In these cases, IFE correction is required, which was the case for the vast majority (97%) of lakes in a nationwide survey (n = 554). For highly absorbing samples, undesirably large diln. factors would be necessary to reduce absorbance below 0.042. For rare EEMs with ATotal > 1.5 (3.0% of the lakes in the Swedish survey), a 2-fold diln. is recommended followed by ABA or CDA correction. This study shows that for the vast majority of natural DOM samples the most commonly applied ABA algorithm provides adequate correction without prior diln.**20**Miller, J. N.; Miller, J. C.*Statistics and Chemometrics for Analytical Chemistry*, 6th ed.; Prentice Hall: Harlow, 2010.Google ScholarThere is no corresponding record for this reference.**21**Brunetti B, D. E. About Estimating the Limit of Detection by the Signal to Noise Approach.*Pharm. Anal. Acta*2015,*6*, 355, DOI: 10.4172/2153-2435.1000355Google ScholarThere is no corresponding record for this reference.

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## References

This article references 21 other publications.

**1**Valeur, B.; Berberan-Santos, M. N.*Molecular Fluorescence: Principles and Applications*, 2nd; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2013.There is no corresponding record for this reference.**2**Lakowicz, J. R. Instrumentation for Fluorescence Spectroscopy.*Principles of Fluorescence Spectroscopy*; Springer US: Boston, MA, 2006; pp 27– 61.There is no corresponding record for this reference.**3**Kimball, J.; Chavez, J.; Ceresa, L.; Kitchner, E.; Nurekeyev, Z.; Doan, H.; Szabelski, M.; Borejdo, J.; Gryczynski, I.; Gryczynski, Z. On the Origin and Correction for Inner Filter Effects in Fluorescence Part I: Primary Inner Filter Effect-the Proper Approach for Sample Absorbance Correction.*Methods Appl. Fluoresc.*2020,*8*, 033002, DOI: 10.1088/2050-6120/ab947c3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1ajsb3J&md5=9c8f9e5cea63b21d8923ee951d011bbfOn the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correctionKimball, Joseph; Chavez, Jose; Ceresa, Luca; Kitchner, Emma; Nurekeyev, Zhangatay; Doan, Hung; Szabelski, Mariusz; Borejdo, Julian; Gryczynski, Ignacy; Gryczynski, ZygmuntMethods and Applications in Fluorescence (2020), 8 (3), 033002CODEN: MAFEB2; ISSN:2050-6120. (IOP Publishing Ltd.)Fluorescence technologies have been the preferred method for detection, anal. sensing, medical diagnostics, biotechnol., imaging, and gene expression for many years. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biol. samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. However, many previous approaches have given only approx. exptl. methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of obsd. results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide. The procedure is generic and applies to many other applications like quantum yield detn., tissue/blood absorption, or acceptor absorption in FRET expts.**4**Panigrahi, S. K.; Mishra, A. K. Study on the Dependence of Fluorescence Intensity on Optical Density of Solutions: The Use of Fluorescence Observation Field for Inner Filter Effect Corrections.*Photochem. Photobiol. Sci.*2019,*18*, 583– 591, DOI: 10.1039/C8PP00498F4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktFymsA%253D%253D&md5=f636a8cc0938a2740da1cedef7973887Study on the dependence of fluorescence intensity on optical density of solutions: the use of fluorescence observation field for inner filter effect correctionsPanigrahi, Suraj Kumar; Mishra, Ashok KumarPhotochemical & Photobiological Sciences (2019), 18 (2), 583-591CODEN: PPSHCB; ISSN:1474-905X. (Royal Society of Chemistry)In this study, we report the identification of absorbance value of an analyte at the excitation wavelength that corresponds to the max. of the obsd. fluorescence intensity obtainable for a certain instrument operating with right-angle fluorescence measurement ([Formula Omitted]). The [Formula Omitted] value depends on the fluorescence observation field (FOF) dimensions of the concerned spectrofluorometer. As the FOF varies from instrument to instrument, this study presents a simple method for obtaining FOF dimensions. Using the knowledge of FOF, absorbance of analyte at the excitation wavelength (Aλex) and emission wavelength (Aλem), we deduced a derived absorbance spectral parameter (Dabs). The obsd. fluorescence intensity of an analyte is proportional to the Dabs. While differentiating Dabs w.r.t. Aλex, the value of [Formula Omitted] for the concentred spectrofluorometer was obtained and subsequently could be used for maximizing fluorescence sensitivity. It was obsd. that when the FOF was a point at the center of a 1 cm path-length cuvette, the ♂[Formula Omitted] value was 0.87 with a progressive widening of FOF, the ♂[Formula Omitted] value increased gradually till ∼1.0. The proposed methodol. was established using two well-known inner filter effect (IFE) correction models (Parker and Lakowicz model). The Dabs obtained from the Parker model corresponded well with the obsd. fluorescence data; however, the Dabs obtained using the Lakowicz model overestimated the loss of fluorescence because of IFE. Using equations derived from the Parker model, the correction of obsd. fluorescence intensity for IFE could be achieved. Furthermore, it is demonstrated that the commonly used the Lakowicz model loses its correction efficiency at absorbance values of ≥0.7.**5**Fonin, A. V.; Sulatskaya, A. I.; Kuznetsova, I. M.; Turoverov, K. K. Fluorescence of Dyes in Solutions with High Absorbance. Inner Filter Effect Correction.*PLoS One*2014,*9*, e103878 DOI: 10.1371/journal.pone.01038785https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1eqt7zN&md5=1465d55a4ac805934a9bf9253e2bb8cfFluorescence of dyes in solutions with high absorbance. Inner filter effect correctionFonin, Alexander V.; Sulatskaya, Anna I.; Kuznetsova, Irina M.; Turoverov, Konstantin K.PLoS One (2014), 9 (7), e103878/1-e103878/8, 8 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Fluorescence is a proven tool in all fields of knowledge, including biol. and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concn. that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dil. solns. We showed that correction must be performed always. Furthermore, high-concn. solns. (high absorbance) are inherent condition in studying of the photophys. properties of fluorescent dyes and the functionally significant interactions of biol. macromols. We proposed an easy to use method to correct the exptl. recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye mols. do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of exptl. detected total fluorescence intensity of the dye on its absorbance coincides with the calcd. dependence and the correction factor for eliminating the primary inner filter effect can be calcd. on the basis of soln. absorbance. It was exptl. shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and addnl. spectral anal. allow to conclude that the most probable reason of the deviation of exptl. detected fluorescence intensity dependence on soln. absorbance from the calcd. dependence is the dye mols. self-quenching, which accompanies resonance radiationless excitation energy transfer.**6**Ma, C.; Li, L.; Gu, J.; Zhu, C.; Chen, G. Inflection Point of the Fluorescence Excitation Spectra Induced by Secondary Inner Filter Effect.*Spectrosc. Lett.*2018,*51*, 319– 323, DOI: 10.1080/00387010.2018.14616556https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1ygurbF&md5=eba2a4f0d8ff4ea325a173fbfda4564bInflection point of the fluorescence excitation spectra induced by secondary inner filter effectMa, Chaoqun; Li, Lei; Gu, Jiao; Zhu, Chun; Chen, GuoqingSpectroscopy Letters (2018), 51 (7), 319-323CODEN: SPLEBX; ISSN:0038-7010. (Taylor & Francis, Inc.)The secondary inner filter effect on the fluorescence excitation spectra of rhodamine 6G aq. solns. with concn. was demonstrated in this paper. The peak of fluorescence excitation spectrum stays at 525 nm at low concns., while it breaks up and turns into valley at high concns. The threshold concn. was detd. to be 3.16 mg/L by the second deriv. spectroscopy. A math. model was proposed to explain the inflection point of fluorescence excitation spectrum caused by the secondary inner filter effect. On the basis of it, the threshold concn. was calcd. to be 2.86 mg/L, approaching to the exptl. result.**7**Panigrahi, S. K.; Mishra, A. K. Inner Filter Effect in Fluorescence Spectroscopy: As a Problem and as a Solution.*J. Photochem. Photobiol., C*2019,*41*, 100318, DOI: 10.1016/j.jphotochemrev.2019.100318There is no corresponding record for this reference.**8**Wang, T.; Zeng, L.-H.; Li, D.-L. A Review on the Methods for Correcting the Fluorescence Inner-Filter Effect of Fluorescence Spectrum.*Appl. Spectrosc. Rev.*2017,*52*, 883– 908, DOI: 10.1080/05704928.2017.13457588https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1OmtrbM&md5=9908fcde03ff0b1bf840393460767b5fA review on the methods for correcting the fluorescence inner-filter effect of fluorescence spectrumWang, Tan; Zeng, Li-Hua; Li, Dao-LiangApplied Spectroscopy Reviews (2017), 52 (10), 883-908CODEN: APSRBB; ISSN:0570-4928. (Taylor & Francis, Inc.)Fluorescence spectroscopy is frequently used to analyze the concn. of fluorescent materials in soln. However, in conventional fluorescence spectroscopy, the response between the fluorescence intensity and fluorophore concn. is nonlinear at high concns. due to uncompensated inner-filter effects (IFE). Many methods to resolve this problem have been developed in recent decades. This review introduces the methods used to correct the IFE, including direct correction and parameter correction. Relevant detection parameters, including the materials, matrixes, detection limits, detection instruments and relative std. deviations, are tabulated. The advantages and limitations of these correction techniques are also discussed. Finally, the methods used to correct for the IFE are summarized, and future research directions are discussed.**9**Kubista, M.; Sjöback, R.; Eriksson, S.; Albinsson, B. Experimental Correction for the Inner-Filter Effect in Fluorescence Spectra.*Analyst*1994,*119*, 417– 419, DOI: 10.1039/AN99419004179https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXivV2ju7w%253D&md5=8eb5dd1c01b9239ef96a3a2700d5cb60Experimental correction for the inner-filter effect in fluorescence spectraKubista, Mikael; Sjoback, Robert; Eriksson, Svante; Albinsson, BoAnalyst (Cambridge, United Kingdom) (1994), 119 (3), 417-19CODEN: ANALAO; ISSN:0003-2654.Recorded fluorescence intensity is in general not proportional to sample concn. owing to absorption of the incident and emitted light passing through the sample to and from the point inside the cell where the emission is detected. This well known inner-filter effect depends on sample absorption and on instrument geometry, and is usually significant even in samples with rather low absorption (the error is about 8% at an absorbance of 0.06 in a 1 cm square cell). In this work the authors show that a particular exptl. set-up can be calibrated for the inner-filter effect from the absorption and fluorescence excitation spectra of a suitable std. The calibration takes only a few minutes and provides correction with sufficient accuracy for most practical situations.**10**Lutz, H.-P.; Luisi, P. L. Correction for Inner Filter Effects in Fluorescence Spectroscopy.*Helv. Chim. Acta*1983,*66*, 1929– 1935, DOI: 10.1002/hlca.1983066070410https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXmt1Wls78%253D&md5=a37690e4d357b4bd6f1e85e40c77edfeCorrection for inner filter effects in fluorescence spectroscopyLutz, Hans Peter; Luisi, Pier LuigiHelvetica Chimica Acta (1983), 66 (7), 1929-35CODEN: HCACAV; ISSN:0018-019X.A method is proposed for correcting exptl. fluorescence readings for inner filter effects, i.e., the absorption of the exciting light and/or the absorption of the emitted radiation, which cause the nonlinearity between fluorescence intensity and fluorophor concn. Basically the method consists of measuring the fluorescence intensity at 2 different points along the diagonal in the cell. Unlike similar methods proposed in the literature, the 2 points are cor. simultaneously for both absorption of excitation and of emission radiation without the necessity of reading the optical d. of the soln., and with a very simple data elaboration.**11**Puchalski, M. M.; Morra, M. J.; von Wandruszka, R. Assessment of Inner Filter Effect Corrections in Fluorimetry.*Fresenius. J. Anal. Chem.*1991,*340*, 341– 344, DOI: 10.1007/BF0032157811https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXlsV2ns7s%253D&md5=6bcb13045993d63a5b79e46c78d417e4Assessment of inner filter effect corrections in fluorimetryPuchalski, M. M.; Morra, M. J.; Von Wandruszka, R.Fresenius' Journal of Analytical Chemistry (1991), 340 (6), 341-4CODEN: FJACES; ISSN:0937-0633.The inner filter effect (IFE) in fluorescence spectroscopy is not easily distinguished from dynamic and static quenching phenomena, since IFE rarely occurs without quenching. IFE corrections may be subject to under- or over-compensation effects that are difficult to assess accurately. To evaluate existing IFE correction procedures, it is proposed that the linearity of resulting Stern-Volmer plots and the relative change of their slopes with temp. be adopted as criteria. Three correction methods are assayed in this manner, and the equation described by T. Gauthier et al. (1986) is found to produce the best results.**12**Kasparek, A.; Smyk, B. A New Approach to the Old Problem: Inner Filter Effect Type I and II in Fluorescence.*Spectrochim. Acta, Part A*2018,*198*, 297– 303, DOI: 10.1016/j.saa.2018.03.02712https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXlt1Sgs7k%253D&md5=cad0341182ea4ab2108c638e2d23ec04A new approach to the old problem: Inner filter effect type I and II in fluorescenceKasparek, Adam; Smyk, BogdanSpectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy (2018), 198 (), 297-303CODEN: SAMCAS; ISSN:1386-1425. (Elsevier B.V.)The fluorescence technique is very popular and has been used in many fields of research. It is simple in its assumptions but not very easy to use. One of the main problems is the inner filter effect (IF) I and II which takes place in the cuvette. IF type I is permanently present, but IF type II occurs only when absorption and fluorescence spectra overlap. To avoid IF type I, absorbencies in the cuvette should be smaller than 0.05, which is however very difficult to obtain in many expts. The authors propose a new method to solve these problems in the case of a Cary Eclipse fluorometer, having horizontally-oriented slits, based on old equations developed in the middle of the last century. This method can be applied for other instruments, even these with vertically-oriented beams, because the authors share scripts written in MATLAB and GRAMS/AI environment. Calcns. in the authors' method enable specifying beam geometry parameters in the cuvette, which is necessary to obtain the correct shape and fluorescence intensity of emission and excitation spectra. Such a specific fluorescence intensity dependence on absorbance can, in many cases, afford possibilities to det. the quantum yield (QY) using slopes of the straight-lines, which was demonstrated using Tryptophan (Trp), Tyrosine (Tyr), and Rhodamine B (RhB) solns. For example, assuming that QY = 0.14 for Tyr, the QY detd. for RhB reached QY = 0.71 ± 0.05, although the measurement for Tyr and RhB was performed at a completely different spectral range.**13**Gu, Q.; Kenny, J. E. Improvement of Inner Filter Effect Correction Based on Determination of Effective Geometric Parameters Using a Conventional Fluorimeter.*Anal. Chem.*2009,*81*, 420– 426, DOI: 10.1021/ac801676j13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVOnsLrN&md5=08dff6ebf7830a79bb2eea9afc7e58a3Improvement of Inner Filter Effect Correction Based on Determination of Effective Geometric Parameters Using a Conventional FluorimeterGu, Qun; Kenny, Jonathan E.Analytical Chemistry (Washington, DC, United States) (2009), 81 (1), 420-426CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The most widely used correction of fluorescence intensities for inner filter effects in conventional (90°) fluorimeters fails at high absorbance values. We have critically examd. this failure, which is caused by the difference between the geometrical parameters (GPs) of the excitation and emission beams in the typical instrument (focused beams) and in the theor. picture on which the correction is based (collimated beams). We provide two types of exptl. measurement of GPs and show that their substitution in the correction equations leads to significant improvements in the linear range of cor. fluorescence. We also demonstrate that math. optimizations give greater improvements and that the optimizations yield GPs consistent with exptl. measurements. For solns. exhibiting primary inner filter effect only, we have extended the range of linearity of cor. fluorescence to aex (absorbance per cm) up to 5.3; for systems with both primary and secondary inner filter effects we have achieved linearity for aex + aem = 6.7. In all cases linear fits have slopes which agree well with the dil. limit. Different series of one- and two-solute solns. were used to demonstrate effectiveness of our correction methods. We also provide a rationale for the unexpected independence of GPs on excitation and emission bandwidths.**14**Tucker, S. A.; Amszi, V. L.; Acree, W. E. Primary and Secondary Inner Filtering. Effect of K2Cr2O7 on Fluorescence Emission Intensities of Quinine Sulfate.*J. Chem. Educ.*1992,*69*, A8, DOI: 10.1021/ed069pA814https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xht1Oktrs%253D&md5=ac3843f19ffd2848d89269aa6eb0123cPrimary and secondary inner filtering: effect of potassium dichromate on fluorescence emission intensities of quinine sulfateTucker, Sheryl A.; Amszi, Vicki L.; Acree, William E., Jr.Journal of Chemical Education (1992), 69 (1), A8, A11-A12CODEN: JCEDA8; ISSN:0021-9584.A lab. expt. is described in which the effect of primary and secondary inner filtering is illustrated by measuring the fluorescence emission signals at 450 nm of 4 ppm quinine sulfate solns. contg. 0, 0.025, 0.05, 0.1, 0.15, and 0.2 mg/mL of K2Cr2O7.**15**Šakić, D.; Weitner, T.; Friganović, T. NINFE. https://ninfe.science/.There is no corresponding record for this reference.**16**Weitner, T.; Friganović, T.; Šakić, D.*Inner Filter Effect Correction for Fluorescence Measurements in Microplates*, 2022, DOI: 10.5281/zenodo.5849302 .There is no corresponding record for this reference.**17**Holland, J. F.; Teets, R. E.; Kelly, P. M.; Timnick, A. Correction of Right-Angle Fluorescence Measurements for the Absorption of Excitation Radiation.*Anal. Chem.*1977,*49*, 706– 710, DOI: 10.1021/ac50014a01117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2sXhsl2rsrc%253D&md5=33b521b8ff03ca7dc5fd3a12e164750aCorrection of right-angle fluorescence measurements for the absorption of excitation radiationHolland, John F.; Teets, Richard E.; Kelly, Patrick M.; Timnick, AndrewAnalytical Chemistry (1977), 49 (6), 706-10CODEN: ANCHAM; ISSN:0003-2700.The effect of excitation beam absorption on measured values of fluorescence was studied with a computer-centered spectrofluorimeter capable of measuring fluorescence and absorbance simultaneously. This effect appears to be independent of the nature of the absorbing species and the excitation and emission wavelengths. A model is proposed and tested which corrects fluorescence, obsd. at 90°, for the attenuation of the excitation beam caused by the absorbance of the fluorophore and any chromophores present in the cell. The resulting absorption-cor. fluorescence is linear with the concn. of the fluorophore in solns. with total absorbances as high as 2.0.**18**Miller, J. N. Inner Filter Effects, Sample Cells and Their Geometry in Fluorescence Spectrometry. In*Standards in Fluorescence Spectrometry: Ultraviolet Spectrometry Group*; Miller, J. N., Ed.; Techniques in Visible and Ultraviolet Spectometry; Springer Netherlands: Dordrecht, 1981; pp 27– 43.There is no corresponding record for this reference.**19**Kothawala, D. N.; Murphy, K. R.; Stedmon, C. A.; Weyhenmeyer, G. A.; Tranvik, L. J. Inner Filter Correction of Dissolved Organic Matter Fluorescence.*Limnol. Oceanogr.: Methods*2013,*11*, 616– 630, DOI: 10.4319/lom.2013.11.61619https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVWhsr7M&md5=e2707da6f3d995ef3c45b15a43ef0658Inner filter correction of dissolved organic matter fluorescenceKothawala, Dolly N.; Murphy, Kathleen R.; Stedmon, Colin A.; Weyhenmeyer, Gesa A.; Tranvik, Lars J.Limnology and Oceanography: Methods (2013), 11 (Nov.), 616-630, 15 pp.CODEN: LOMIBY; ISSN:1541-5856. (American Society of Limnology and Oceanography)The fluorescence of dissolved org. matter (DOM) is suppressed by a phenomenon of self-quenching known as the inner filter effect (IFE). Despite widespread use of fluorescence to characterize DOM in surface waters, the advantages and constraints of IFE correction are poorly defined. We assessed the effectiveness of a commonly used absorbance-based approach (ABA), and a recently proposed controlled diln. approach (CDA) to correct for IFE. Linearity between cor. fluorescence and total absorbance (ATotal; the sum of absorbance at excitation and emission wavelengths) across the full excitation-emission matrix (EEM) in diln. series of four samples indicated both ABA and CDA were effective to an absorbance of at least 1.5 in a 1 cm cell, regardless of wavelength positioning. In regions of the EEMs where signal to background noise (S/N) was low, CDA correction resulted in more variability than ABA correction. From the ABA algorithm, the onset of significant IFE (>5%) occurs when absorbance exceeds 0.042. In these cases, IFE correction is required, which was the case for the vast majority (97%) of lakes in a nationwide survey (n = 554). For highly absorbing samples, undesirably large diln. factors would be necessary to reduce absorbance below 0.042. For rare EEMs with ATotal > 1.5 (3.0% of the lakes in the Swedish survey), a 2-fold diln. is recommended followed by ABA or CDA correction. This study shows that for the vast majority of natural DOM samples the most commonly applied ABA algorithm provides adequate correction without prior diln.**20**Miller, J. N.; Miller, J. C.*Statistics and Chemometrics for Analytical Chemistry*, 6th ed.; Prentice Hall: Harlow, 2010.There is no corresponding record for this reference.**21**Brunetti B, D. E. About Estimating the Limit of Detection by the Signal to Noise Approach.*Pharm. Anal. Acta*2015,*6*, 355, DOI: 10.4172/2153-2435.1000355There is no corresponding record for this reference.

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