Fluorescent proteins for fluctuation spectroscopy in vivo.

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Fast and transient molecular interactions like receptor ligand binding, membrane domain formation and dissipation, or transcription factor assembly on DNA are crucial components of cellular physiology and homeostasis. Fascinatingly, such short-lived, nano-scale interactions determine signaling responses, differentiation and development, dynamic processes spanning a wide range of time- and length-scales. Therefore, quantification of these molecular dynamics and interactions would allow us to eavesdrop on the fundamental processes underlying life. However, it is challenging to directly follow these dynamics using conventional imaging methods such as a confocal microscope due to slow acquisition times and the transient nature of single molecule events. Fluorescence fluctuations spectroscopy (FFS) methods have been used as a remedy for decades. While immensely powerful in vitro, FFS has only rarely been applied in vivo probably due to the challenges with low signal-to-noise ratios, high background fluorescence, and optical aberrations in complex samples. Here, we explore the use of common fluorescent proteins (FPs) to study diffusion dynamics in live zebrafish embryos and compare the signal quality with a tissue culture model. We show that the main drivers for high-quality FFS acquisitions are molecular brightness and photostability of the FPs over a wide range of expression levels. In combination with numerical simulations, we estimate regimes where data can be confidently acquired. Finally, we present our recommendations on labelling proteins of interest for in vitro and in vivo studies.