Figure 1.
Rectangular and triangular two-dimensional spectra.
(A) Example of a conventional array of excitation/emission wavelengths used for acquisition of two-dimensional fluorescence spectra. The diagonal (λem = λex) is shown with the open symbols. The conventional 1D emission spectrum is a horizontal set of points for each excitation wavelength to the right of the open symbol. Correspondingly, the 1D excitation spectrum is a vertical set of points below the open symbol. (B) The triangular array of excitation/emission wavelengths where the emission ranges always begin at a greater wavelength than the excitation wavelength in each row. The specific 100 nm increment in this figure is chosen solely for clarity of presentation.
Figure 2.
A typical workflow of the two-dimensional acquisition and data analysis.
Figure 3.
Manipulation of the graphic output of Fluorescence2D.
The interactive tools of MATLAB allow for easy adjustment, inspection, and export of the graphics created by Fluorescence2D. In the panel A, arrows indicate locations of the zoom tool (1) used to enlarge the desired area, the data inspector tool (2) allowing to determine values of wavelength and intensity at any specific point, and the interactive graphics mode toggle (3) for adjustment of font sizes, labels, ticks, and colors. Export of the final image into a desired format is performed through File:Save As menu item as shown in Panel B.
Figure 4.
Two-dimensional spectra of H-Ras in the complex with mant-GDP.
(Panel A) The 2D spectrum of the buffer solution (without baseline subtraction) is shown contoured between 0 and 10% of total intensity (blue to red). The first order reflection, 1; the second order reflection, 2; Raman scattering, 3. The black solid line separates the lower area with experimental data from the upper area added to make the dataset rectangular for visualization. (Panel B) The 2D spectrum of H-Ras in the complex with mant-GDP. The spectrum of the buffer solution (from Panel A) was subtracted as a baseline; the resulting graph was contoured from 3 to 13% of total intensity scale. Tyrosine fluorescence, 4; its second order reflection, 5; mant-GDP emission, 6 and 7. Spectra in both panels were “zoomed in” using interactive MATLAB graphics tools. The original and processed experimental data along with the corresponding fluorescence2Dmain.m script are included with the Fluorescence2D package in the sample_data/folder.
Figure 5.
One-dimensional slices of the 2D spectrum extracted at specified emission and excitation wavelengths.
The 1D spectra were extracted from the full 2D spectrum at the excitation wavelength of 360(A), and at the emission wavelength of 440 nm (B), respectively.