5 research outputs found
Transgenic mice encoding modern imaging probes : Properties and applications
Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.Peer reviewe
Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium Indicator
Near-infrared (NIR) genetically encoded calcium indicators (GECIs) are becoming powerful tools for neuroscience. Because of their spectral characteristics, the use of NIR GECIs helps to avoid signal loss from the absorption by body pigments, light-scattering, and autofluorescence in mammalian tissues. In addition, NIR GECIs do not suffer from cross-excitation artifacts when used with common fluorescent indicators and optogenetics actuators. Although several NIR GECIs have been developed, there is no NIR GECI currently available that would combine the high brightness in cells and photostability with small size and fast response kinetics. Here, we report a small FRET-based NIR fluorescent calcium indicator iGECInano. We characterize iGECInano in vitro, in non-neuronal mammalian cells, and primary mouse neurons. iGECInano demonstrates the improvement in the signal-to-noise ratio and response kinetics compared to other NIR GECIs.Peer reviewe
Optogenetic manipulation and photoacoustic imaging using a near-infrared transgenic mouse model
Optogenetic manipulation and optical imaging in the near-infrared range allow non-invasive light-control and readout of cellular and organismal processes in deep tissues in vivo. Here, we exploit the advantages of Rhodopseudomonas palustris BphP1 bacterial phytochrome, which incorporates biliverdin chromophore and reversibly photoswitches between the ground (740-800 nm) and activated (620-680 nm) states, to generate a loxP-BphP1 transgenic mouse model. The mouse enables Cre-dependent temporal and spatial targeting of BphP1 expression in vivo. We validate the optogenetic performance of endogenous BphP1, which in the activated state binds its engineered protein partner QPAS1, to trigger gene transcription in primary cells and living mice. We demonstrate photoacoustic tomography of BphP1 expression in different organs, developing embryos, virus-infected tissues and regenerating livers, with the centimeter penetration depth. The transgenic mouse model provides opportunities for both near-infrared optogenetics and photoacoustic imaging in vivo and serves as a source of primary cells and tissues with genomically encoded BphP1. Optogenetic tools can be used as in vivo imaging probes. Here the authors generate a loxP-BphP1 transgenic mouse to enable Cre-dependent temporal and spatial targeting of BphP1 expression in vivo; they show photoacoustic tomography of BphP1 expression in developing embryos and regenerating livers.Peer reviewe
On the importance of low-frequency signals in functional and molecular photoacoustic computed tomography
In photoacoustic computed tomography (PACT) with short-pulsed laser
excitation, wideband acoustic signals are generated in biological tissues with
frequencies related to the effective shapes and sizes of the optically
absorbing targets. Low-frequency photoacoustic signal components correspond to
slowly varying spatial features and are often omitted during imaging due to the
limited detection bandwidth of the ultrasound transducer, or during image
reconstruction as undesired background that degrades image contrast. Here we
demonstrate that low-frequency photoacoustic signals, in fact, contain
functional and molecular information, and can be used to enhance structural
visibility, improve quantitative accuracy, and reduce spare-sampling artifacts.
We provide an in-depth theoretical analysis of low-frequency signals in PACT,
and experimentally evaluate their impact on several representative PACT
applications, such as mapping temperature in photothermal treatment, measuring
blood oxygenation in a hypoxia challenge, and detecting photoswitchable
molecular probes in deep organs. Our results strongly suggest that
low-frequency signals are important for functional and molecular PACT