Photoacoustic tomography (PAT) of genetically encoded probes allows imaging of targeted biological processes with high spatial resolution at depths. Here, we combined multi-scale photoacoustic imaging with, for the first time, a reversibly switchable non-fluorescent bacterial phytochrome BphP1. With a heme-derived biliverdin chromophore, BphP1 has the most red-shifted absorption among reported genetically encoded probes, and is reversibly photoconvertible between its red and near-infrared light absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, enabling differential imaging that substantially removed background signals, enhanced detection sensitivity, increased penetration depth, and improved spatial resolution. In doing so, we monitored tumor growth and metastasis with a ~100 µm resolution at depths approaching 10 mm using photoacoustic computed tomography, and imaged individual cancer cells with a sub-optical-diffraction resolution of ~140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at different length scales.

Light-mediated control of protein-protein interactions to regulate metabolic pathways is an important approach of optogenetics. Here, we report the first optogenetic system based on a reversible light-induced binding between a bacterial phytochrome BphP1 and its natural partner PpsR2 from Rhodopseudomonas palustris bacteria. We extensively characterized the BphP1–PpsR2 interaction both in vitro and in mammalian cells, and then used it to translocate target proteins to specific cellular compartments, such as plasma membrane and nucleus. Applying this approach we achieved a light-control of cell morphology resulting in the substantial increase of cell area. We next demonstrated the light-induced gene expression with the 40-fold contrast in cultured cells, 32-fold subcutaneously and 5.7-fold in deep tissues in mice. The unique characteristics of the BphP1–PpsR2 optogenetic system are its sensitivity to 740–780 nm near-infrared light, ability to utilize an endogenous biliverdin chromophore in eukaryotes including mammals, and spectral compatibility with blue-light optogenetic systems.

Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein–like probes, including near-infrared fluorescence, independence of oxygen, small size, and photo-sensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells.

Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. However, available NIR optogenetic systems consist of several protein components of large size and multidomain structure. Here, we engineer single-component NIR systems consisting of evolved photosensory core module of Idiomarina sp. bacterial phytochrome, named iLight, which are smaller and packable in adeno-associated virus. We characterize iLight in vitro and in gene transcription repression in bacterial and gene transcription activation in mammalian cells. Bacterial iLight system shows 115-fold repression of protein production. Comparing to multi-component NIR systems, mammalian iLight system exhibits higher activation of 65-fold in cells and faster 6-fold activation in deep tissues of mice. Neurons transduced with viral-encoded iLight system exhibit 50-fold induction of fluorescent reporter. NIR light-induced neuronal expression of green-light-activatable CheRiff channelrhodopsin causes 20-fold increase of photocurrent and demonstrates efficient spectral multiplexing.

Near-infrared (NIR, 740-780 nm) optogenetic systems are well-suited to spectral multiplexing with blue-light-controlled tools. Here, we present two protocols, one for regulation of gene transcription and another for control of protein localization, that use a NIR-responsive bacterial phytochrome BphP1-QPAS1 optogenetic pair. In the first protocol, cells are transfected with the optogenetic constructs for independently controlling gene transcription by NIR (BphP1-QPAS1) and blue (LightOn) light. The NIR and blue-light-controlled gene transcription systems show minimal spectral crosstalk and induce a 35- to 40-fold increase in reporter gene expression. In the second protocol, the BphP1-QPAS1 pair is combined with a light-oxygen-voltage-sensing (LOV) domain-based construct into a single optogenetic tool, termed iRIS. This dual-light-controllable protein localization tool allows tridirectional protein translocation among the cytoplasm, nucleus and plasma membrane. Both procedures can be performed within 3-5 d. Use of NIR light-controlled optogenetic systems should advance basic and biomedical research.