Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a nonfluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, and an adjustment of the equilibrium poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows one to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell-stimulated emission depletion (STED) microscopy or a spontaneously blinking dye for single-molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

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Phycoerythrin is the major light-harvesting pigment protein in red algae and is nowadays widely used as a fluorescent probe in biotechnological applications such as flow cytometry and immunofluorescence microscopy. In addition, it has had substantial economic impact due to its potential as a natural food colorant. However, knowledge on the precise molecular composition of phycoerythrin is limited. Here, we use a combination of high-resolution native mass spectrometry (MS) and fluorescence spectroscopy to characterize the assembly properties of the B-phycoerythrin protein complex from Porphyridium cruentum. Our data highlight the stabilizing role of the c subunit in the intact B-phycoerythrin protein complex. In addition, by native MS we monitor B-phycoerythrin (dis)assembly intermediates, providing insight into which species contribute to B-phycoerythrins color and the factors that give B-phycoerythrin its highly fluorescent properties. Together, the data provide significant insights into the structural properties of B-phycoerythrin which is beneficial for its use within the biotechnology industry.