Improving the spectral resolution in fluorescence microscopy through shaped-excitation imaging

The visualization of distinct molecular species represents an important challenge of bio-imaging research. In past decades, the development of multicolor fluorescent (FL) labels has greatly improved our ability to track biological analytes, paving the way for important advances in understanding the cell dynamics. It remains challenging, however, to visualize a large number of different fluorephores simultaneously. Owing to a spectrally broad absorption of fluorescent dyes, only up to five color categories can be resolved at once. Here, we demonstrate a general strategy for distinguishing between multiple fluorescent targets in acquired microscopy images with improved accuracy. The present strategy is enabled through spectral shaping of the excitation light with an optical filter that uniquely attenuates the light absorption of each fluorophore in the investigated sample. The resulting emission changes, induced by such excitation modulation, are therefore target-specific and can be used for identifying various fluorescent species. The technique is demonstrated through an accurate identification of 8 different CdSe dyes with absorption maxima spanning the 520-620 spectral range. It is subsequently applied for accurate measurements of the pH balance in buffers emulating a metabolism of tumor cells.