Near-infrared PAINT localization microscopy via chromophore replenishment of phytochrome-derived fluorescent tag.

Bacterial phytochromes are attractive molecular templates for engineering fluorescent proteins (FPs) because their near-infrared (NIR) emission significantly extends the spectral coverage of GFP-like FPs. Existing phytochrome-based FPs covalently bind heme-derived tetrapyrrole chromophores and exhibit constitutive fluorescence. Here we introduce Rep-miRFP, an NIR imaging probe derived from bacterial phytochrome, which interacts non-covalently and reversibly with biliverdin chromophore. In Rep-miRFP, the photobleached non-covalent adduct can be replenished with fresh biliverdin, restoring fluorescence. By exploiting this chromophore renewal capability, we demonstrate NIR PAINT nanoscopy in mammalian cells using Rep-miRFP.

Characterization of two near-infrared genetically encoded voltage indicators.

Significance: Efforts starting more than 20 years ago led to increasingly well performing genetically encoded voltage indicators (GEVIs) for optical imaging at wavelengths <600 nm. Although optical imaging in the >600 nm wavelength range has many advantages over shorter wavelength approaches for mesoscopic in vivo monitoring of neuronal activity in the mammalian brain, the availability and evaluation of well performing near-infrared GEVIs are still limited. Aim: Here, we characterized two recent near-infrared GEVIs, Archon1 and nirButterfly, to support interested tool users in selecting a suitable near-infrared GEVI for their specific research question requirements. Approach: We characterized side-by-side the brightness, sensitivity, and kinetics of both near-infrared GEVIs in a setting focused on population imaging. Results: We found that nirButterfly shows seven-fold higher brightness than Archon1 under the same conditions and faster kinetics than Archon1 for population imaging without cellular resolution. But Archon1 showed larger signals than nirButterfly. Conclusions: Neither GEVI characterized here surpasses in all three key parameters (brightness, kinetics, and sensitivity), so there is no unequivocal preference for one of the two. Our side-by-side characterization presented here provides new information for future in vitro and ex vivo experimental designs.

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms.

Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400-650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.

Near-Infrared Optogenetic Module for Conditional Protein Splicing.

Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue penetration, holds particular promise. However, the optogenetic control of polypeptide bond formation has not yet been developed. In this study, we introduce a NIR optogenetic module for conditional protein splicing (CPS) based on the gp41-1 intein. We optimized the module to minimize background signals in the darkness and to maximize the contrast between light and dark conditions. Next, we engineered a NIR CPS gene expression system based on the protein ligation of a transcription factor. We applied the NIR CPS for light-triggered protein cleavage to activate gasdermin D, a pore-forming protein that induces pyroptotic cell death. Our NIR CPS optogenetic module represents a promising tool for controlling molecular processes through covalent protein linkage and cleavage.

Photodegradable by Yellow-Orange Light degFusionRed Optogenetic Module with Autocatalytically Formed Chromophore.

Optogenetic systems driven by yellow-orange light are required for the simultaneous regulation of several cellular processes. We have engineered the red fluorescent protein FusionRed into a 26 kDa monomeric optogenetic module, called degFusionRed. Unlike other fluorescent protein-based optogenetic domains, which exhibit light-induced self-inactivation by generating reactive oxygen species, degFusionRed undergoes proteasomal degradation upon illumination with 567 nm light. Similarly to the parent protein, degFusionRed has minimal absorbance at 450 nm and above 650 nm, making it spectrally compatible with blue and near-infrared-light-controlled optogenetic tools. The autocatalytically formed chromophore provides degFusionRed with an additional advantage over most optogenetic tools that require the binding of the exogenous chromophores, the amount of which varies in different cells. The degFusionRed efficiently performed in the engineered light-controlled transcription factor and in the targeted photodegradation of the protein of interest, demonstrating its versatility as the optogenetic module of choice for spectral multiplexed interrogation of various cellular processes.

Nuclear Localization Signals for Optimization of Genetically Encoded Tools in Neurons.

Nuclear transport in neurons differs from that in non-neuronal cells. Here we developed a non-opsin optogenetic tool (OT) for the nuclear export of a protein of interest induced by near-infrared (NIR) light. In darkness, nuclear import reverses the OT action. We used this tool for comparative analysis of nuclear transport dynamics mediated by nuclear localization signals (NLSs) with different importin specificities. We found that widely used KPNA2-binding NLSs, such as Myc and SV40, are suboptimal in neurons. We identified uncommon NLSs mediating fast nuclear import and demonstrated that the performance of the OT for nuclear export can be adjusted by varying NLSs. Using these NLSs, we optimized the NIR OT for light-controlled gene expression for lower background and higher contrast in neurons. The selected NLSs binding importins abundant in neurons could improve performance of genetically encoded tools in these cells, including OTs and gene-editing tools.

Technologies for large-scale mapping of functional neural circuits active during a user-defined time window.

The mapping of neural circuits activated during behavior down to individual neurons is crucial for decoding how the brain processes information. Technologies allowing activity-dependent labeling of neurons during user-defined restricted time windows are rapidly developing. Precise marking of the time window with light, in addition to chemicals, is now possible. In these technologies, genetically encoded molecules integrate molecular events resulting from neuronal activity with light/drug-dependent events. The outputs are either changes in fluorescence or activation of gene expression. Molecular reporters allow labeling of activated neurons for visualization and cell-type identification. The transcriptional readout also allows further control of activated neuronal populations using optogenetic tools as reporters. Here we review the design of these technologies and discuss their demonstrated applications to reveal previously unknown connections in the mammalian brain. We also consider the strengths and weaknesses of the current approaches and provide a perspective for the future.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

Multiscale Photoacoustic Tomography of a Genetically Encoded Near-Infrared FRET Biosensor.

Photoacoustic tomography (PAT) with genetically encoded near-infrared probes enables visualization of specific cell populations in vivo at high resolution deeply in biological tissues. However, because of a lack of proper probes, PAT of cellular dynamics remains unexplored. Here, the authors report a near-infrared Forster resonance energy transfer (FRET) biosensor based on a miRFP670-iRFP720 pair of the near-infrared fluorescent proteins, which enables dynamic functional imaging of active biological processes in deep tissues. By photoacoustically detecting the changes in the optical absorption of the miRFP670 FRET-donor, they monitored cell apoptosis in deep tissue at high spatiotemporal resolution using PAT. Specifically, they detected apoptosis in single cells at a resolution of ≈3 µm in a mouse ear tumor, and in deep brain tumors (>3 mm beneath the scalp) of living mice at a spatial resolution of ≈150 µm with a 20 Hz frame rate. These results open the way for high-resolution photoacoustic imaging of dynamic biological processes in deep tissues using NIR biosensors and PAT.

Near-infrared and far-red genetically encoded indicators of neuronal activity.

Genetically encoded fluorescent indicators of neuronal activity are ultimately developed to dissect functions of neuronal ensembles during behavior in living animals. Recent development of near-infrared shifted calcium and voltage indicators moved us closer to this goal and enabled crosstalk-free combination with blue light-controlled optogenetic tools for all-optical control and readout. Here I discuss designs of recent near-infrared and far-red calcium and voltage indicators, compare their properties and performance, and overview their applications to spectral multiplexing and in vivo imaging. I also provide perspectives for further development.

A guide to the optogenetic regulation of endogenous molecules.

Genetically encoded tools for the regulation of endogenous molecules (RNA, DNA elements and protein) are needed to study and control biological processes with minimal interference caused by protein overexpression and overactivation of signaling pathways. Here we focus on light-controlled optogenetic tools (OTs) that allow spatiotemporally precise regulation of gene expression and protein function. To control endogenous molecules, OTs combine light-sensing modules from natural photoreceptors with specific protein or nucleic acid binders. We discuss OT designs and group OTs according to the principles of their regulation. We outline characteristics of OT performance, discuss considerations for their use in vivo and review available OTs and their applications in cells and in vivo. Finally, we provide a brief outlook on the development of OTs.

Multiplex Imaging of Rho GTPase Activities in Living Cells.

Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.

Single-component near-infrared optogenetic systems for gene transcription regulation.

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.

Toward photoswitchable electronic pre-resonance stimulated Raman probes.

Reversibly photoswitchable probes allow for a wide variety of optical imaging applications. In particular, photoswitchable fluorescent probes have significantly facilitated the development of super-resolution microscopy. Recently, stimulated Raman scattering (SRS) imaging, a sensitive and chemical-specific optical microscopy, has proven to be a powerful live-cell imaging strategy. Driven by the advances of newly developed Raman probes, in particular the pre-resonance enhanced narrow-band vibrational probes, electronic pre-resonance SRS (epr-SRS) has achieved super-multiplex imaging with sensitivity down to 250 nM and multiplexity up to 24 colors. However, despite the high demand, photoswitchable Raman probes have yet to be developed. Here, we propose a general strategy for devising photoswitchable epr-SRS probes. Toward this goal, we exploit the molecular electronic and vibrational coupling, in which we switch the electronic states of the molecules to four different states to turn their ground-state epr-SRS signals on and off. First, we showed that inducing transitions to both the electronic excited state and triplet state can effectively diminish the SRS peaks. Second, we revealed that the epr-SRS signals can be effectively switched off in red-absorbing organic molecules through light-facilitated transitions to a reduced state. Third, we identified that photoswitchable proteins with near-infrared photoswitchable absorbance, whose states are modulable with their electronic resonances detunable toward and away from the pump photon energy, can function as the photoswitchable epr-SRS probes with desirable sensitivity (<1 µM) and low photofatigue (>40 cycles). These photophysical characterizations and proof-of-concept demonstrations should advance the development of novel photoswitchable Raman probes and open up the unexplored Raman imaging capabilities.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

A near-infrared genetically encoded calcium indicator for in vivo imaging.

While calcium imaging has become a mainstay of modern neuroscience, the spectral properties of current fluorescent calcium indicators limit deep-tissue imaging as well as simultaneous use with other probes. Using two monomeric near-infrared (NIR) fluorescent proteins (FPs), we engineered an NIR Förster resonance energy transfer (FRET)-based genetically encoded calcium indicator (iGECI). iGECI exhibits high levels of brightness and photostability and an increase up to 600% in the fluorescence response to calcium. In dissociated neurons, iGECI detects spontaneous neuronal activity and electrically and optogenetically induced firing. We validated the performance of iGECI up to a depth of almost 400 µm in acute brain slices using one-photon light-sheet imaging. Applying hybrid photoacoustic and fluorescence microscopy, we simultaneously monitored neuronal and hemodynamic activities in the mouse brain through an intact skull, with resolutions of ~3 µm (lateral) and ~25-50 µm (axial). Using two-photon imaging, we detected evoked and spontaneous neuronal activity in the mouse visual cortex, with fluorescence changes of up to 25%. iGECI allows biosensors and optogenetic actuators to be multiplexed without spectral crosstalk.

Screening and Cellular Characterization of Genetically Encoded Voltage Indicators Based on Near-Infrared Fluorescent Proteins.

We developed genetically encoded voltage indicators using a transmembrane voltage-sensing domain and bright near-infrared fluorescent proteins derived from bacterial phytochromes. These new voltage indicators are excited by 640 nm light and emission is measured at 670 nm, allowing imaging in the near-infrared tissue transparency window. The spectral properties of our new indicators permit seamless voltage imaging with simultaneous blue-green light optogenetic actuator activation as well as simultaneous voltage-calcium imaging when paired with green calcium indicators. Iterative optimizations led to a fluorescent probe, here termed nirButterfly, which reliably reports neuronal activities including subthreshold membrane potential depolarization and hyperpolarization as well as spontaneous spiking or electrically- and optogenetically evoked action potentials. This enables largely improved all-optical causal interrogations of physiology.

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales.

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~105 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.

Fluorescent Biosensors for Neurotransmission and Neuromodulation: Engineering and Applications.

Understanding how neuronal activity patterns in the brain correlate with complex behavior is one of the primary goals of modern neuroscience. Chemical transmission is the major way of communication between neurons, however, traditional methods of detection of neurotransmitter and neuromodulator transients in mammalian brain lack spatiotemporal precision. Modern fluorescent biosensors for neurotransmitters and neuromodulators allow monitoring chemical transmission in vivo with millisecond precision and single cell resolution. Changes in the fluorescent biosensor brightness occur upon neurotransmitter binding and can be detected using fiber photometry, stationary microscopy and miniaturized head-mounted microscopes. Biosensors can be expressed in the animal brain using adeno-associated viral vectors, and their cell-specific expression can be achieved with Cre-recombinase expressing animals. Although initially fluorescent biosensors for chemical transmission were represented by glutamate biosensors, nowadays biosensors for GABA, acetylcholine, glycine, norepinephrine, and dopamine are available as well. In this review, we overview functioning principles of existing intensiometric and ratiometric biosensors and provide brief insight into the variety of neurotransmitter-binding proteins from bacteria, plants, and eukaryotes including G-protein coupled receptors, which may serve as neurotransmitter-binding scaffolds. We next describe a workflow for development of neurotransmitter and neuromodulator biosensors. We then discuss advanced setups for functional imaging of neurotransmitter transients in the brain of awake freely moving animals. We conclude by providing application examples of biosensors for the studies of complex behavior with the single-neuron precision.

Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins.

Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness. Here, by combining biochemical, spectroscopic and resonance Raman (RR) assays, we purified and characterized an iRFP variant that contains a BV chromophore simultaneously bound to two cysteines. This protein with the unusual double-Cys attached BV showed the highest fluorescence quantum yield (FQY) of 16.6% reported for NIR FPs, whereas the initial iRFP appeared to be a mixture of species with a mean FQY of 11.1%. The purified protein was also characterized with 1.3-fold higher extinction coefficient that together with FQY resulted in almost two-fold brighter fluorescence than the original iRFP as isolated. This work shows that the high FQY of iRFPs with two cysteines is a direct consequence of the double attachment. The PAS-Cys, GAF-Cys and double-Cys attachment each entails distinct configurational constraints of the BV adduct, which can be identified by distinct RR spectroscopic features, i.e. the marker band including the C=C stretching coordinate of the ring A-B methine bridge, which was previously identified as being characteristic for rigid chromophore embedment and high FQY. Our findings can be used to rationally engineer iRFP variants with enhanced FQYs.