Albert Einstein College of Medicine


Mammalian tissues are the most transparent to light in a near-infrared (NIR) optical transparency window (650-900 nm) where hemologlobin and melanin absorbance decreases, and water absorbance is still low. An optimal fluorescent probe for imaging in tissue should have both excitation and emission spectra located in the NIR range and be genetically encoded. The excitation spectra of far-red shifted fluorescent proteins (FPs) of a green fluorescent protein (GFP) family are completely outside of the NIR region, limiting their use in deep-tissue and whole-body imaging. Attempts to engineer FPs with longer wavelength excitation failed, indicating natural limits of the autocatalytic chromophore system of the GFP-like proteins.

To circumvent these problems, NIR FPs can be engineered from phytochromes. Phytochromes are cytoplasmic photosensory receptors that absorb light in the far-red and NIR parts of spectrum. In these proteins natural linear tetrapyrrole bilins serve as chromophores. These molecules are enzymatic derivatives of heme and include biliverdin, phycocyanobilin and phytochromobilin. Importantly, the bacterial phytochrome photoreceptors (BphPs) covalently bind biliverdin, which is a component of endogenous mammalian heme metabolism. Thus, NIR FPs engineered from BphPs do not require supply of any external cofactors to fluoresce.

To advance in vivo imaging, monitoring and manipulation of intracellular processes we develop novel NIR fluorescent proteins, biosensors and optogenetic tools based on BphPs, and systematically optimize their spectral, photochemical and biophysical properties by utilizing advanced protein engineering and high-throughput screening technologies. Our projects result in the collections of advanced multicolor NIR probes, which are as versatile as the constructs based on the GFP, flavoprotein and rhodopsin protein families. These NIR probes and molecular tools expand the deep-tissue imaging and noninvasive photomanipulating capabilities in living cells, tissues and whole animals.

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