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.