Figure 1.
The dependences of the total fluorescence intensity of N-acetyl tryptophan amide (NATA) on its absorbance.
Curve 1 (solid line) represents the fluorescence intensity that was calculated according to Eq. 2; the circles on the curve represent the fluorescence intensity values recorded by the Cary Eclipse (Agilent Technologies, Australia) spectrofluorimeter. Curves 2 and 3 represent the fluorescence intensity recorded by a homemade spectrofluorimeter [20] and by a Fluorolog-3 (Horiba, Japan) spectrofluorimeter, respectively. The straight line F = 2.303AFLq is tangent to curves 1, 2 and 3 at AFL = 0. Here k′ is chosen so that k′I0 = 1, and consequently numerically equals to q at
. The inset represents the dependence of W on AFL calculated by Eq. 4 (curve 1) and determined experimentally for the homemade spectrofluorimeter (curve 2). For this plot, dyes with different fluorescence quantum yields were used: quinine sulfate (q = 0.52 [36], green circles) and NATA (q = 0.14 [21] red circles).
Figure 2.
The dependence of the total fluorescence intensity of N-acetyl tryptophan amide (NATA) on its absorbance at high concentrations of fluorophore, as recorded by a Cary Eclipse spectrofluorimeter.
Curves 1, 2 and 3 are the same as in Figure 1. Curves 4 and 5 represent the fluorescence intensity recorded with the use of microcells (5×5 mm cross section) and in triangular cell. The absorbance was determined using a Hitachi U-3900H spectrophotometer in cells with different optical path lengths. Inset: The corrected and normalized fluorescence excitation and emission spectra of NATA. The fluorescence excitation and emission spectra were recorded by a Cary Eclipse spectrofluorimeter at λem = 350 nm and λex = 280 nm, respectively. The correction was performed according to Eq. 4. The red, blue and green curves correspond to A280 = 60, 1.5 and 0.2, respectively. The black curve is the absorption spectrum.
Figure 3.
Schematic presentation of the light fluxes in the spectrofluorimeters with vertical (standard) and horizontal slit geometries.
The figure was constructed on the basis of the advertising material by Steensrud [30]. In both cases, the slits are projected to the center of the cell.
Figure 4.
The dependence of the total fluorescence intensity of ATTO-425 on the solution absorbance.
On Panels A and B the red curves F(AFL) and F0(AFL) represent the dependences of the total fluorescence intensity on the solution absorbance calculated according to Eq. 2 and Eq. 3 (q = 0.9), respectively. The dependence of the ATTO-425 fluorescence on its absorbance recorded by a Cary Eclipse spectrofluorimeter is given on Panel A (black curve); this dependence after correction for the primary inner filter effect and for the primary and secondary inner filter effects is given in Panel B (curves 1 and 2, respectively). For comparison, the dependence of the total fluorescence on absorbance for NATA is given by the green curve (q = 0.14 [21]). Insets: Fluorescence spectra of ATTO-425 in solutions with different absorbance values (black curve 1–0.1, 2; dark red curve 2–0.2, dark green curve 3–0.5, dark yellow curve 4–0.7, dark blue curve 5–0.9, dark violet curve 6–2.8, dark blue curve 7–5.7, pink curve 8–9.5, red curve 9–13.9, green curve 10–20.1, blue curve 11–29.4): recorded (Panel A) and corrected for primary inner filter effect (Panel B).
Figure 5.
Correction of the fluorescence of the dye ATTO-425 on the secondary inner filter effect.
The dashed lines show the fluorescence spectra of ATTO-425 in solutions of its different concentration corrected for primary inner filter effect. Solid lines are the fluorescence spectra measured for a solution with absorbance 0.1 (i.e., where the effect of reabsorption of the fluorescence light can be neglected) and normalized so that at a wavelength of 550 nm (where the absorption spectrum and fluorescence spectrun do not overlap) the its intensity coincided with measured fluorescence intensity of solution with given concentration, corrected for the primary inner filter effect. This figure also shows the long wavelength parts of absorption spectra (the left ordinary axis). Colors and numbers on the curves are the same as in Figure 4.
Figure 6.
Normalized absorption and fluorescence spectrum of ATTO-425. Colors and numbers on the curves are the same as in Figure 4.
Figure 7.
Normalized fluorescence excitation spectra of ATTO-425 solutions with different absorbance values before (dotted lines) and after (solid lines) correction for the primary inner filter effect (λem = 510 nm).
Colors and numbers on the curves are the same as in Figure 4. The normalized absorption spectrum is also given (red bold curve).