Near-infrared light is favorable for imaging in mammalian tissues due to its low absorbance by hemoglobin, melanin, and water, as well as reduced light-scattering and phototoxicity for such wavelengths. Therefore, fluorescent proteins and sensors with fluorescence spectra within the “optical window”, characterized by a wavelength between 650 and 1350 nm, are beneficial for in vivo imaging. However, a major limitation in this field is the availability of genetically encoded near-infrared probes optimized for in vivo expression. To overcome this limitation we developed a new way to engineer fluorescent reporter proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that are identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins from a mutant library in just a few hours, evaluating each along multiple performance axes. Using this method, we created two groups of genetically encoded near-infrared fluorescent probes with biochemical and biophysical characteristics optimized for in vivo imaging. The first group of probes is represented by monomeric near-infrared fluorescent proteins exhibiting higher brightness and photostability than previously developed, spectrally similar, near-infrared fluorescent proteins, which could be readily expressed in neurons in culture and in vivo and imaged using both one- and two-photon microscopy. The second group of probes is represented by high-performance opsin-based fluorescent voltage sensors characterized by submillisecond temporal resolution (~0.6 and ~0.1 ms, respectively), high voltage sensitivity (44 and 20% of ΔF/F per 100 mV), and extreme photostability allowing for long-term (&gt;15 min) imaging of neuronal activity with no detectable phototoxicity. We demonstrated the utility of the voltage sensors by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

Genetically encoded near-infrared fluorescent probes for in vivo imaging

256th National Meeting (2018)

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