Video-based pooled screening yields improved far-red genetically encoded voltage indicators.

Video-based screening of pooled libraries is a powerful approach for directed evolution of biosensors because it enables selection along multiple dimensions simultaneously from large libraries. Here we develop a screening platform, Photopick, which achieves precise phenotype-activated photoselection over a large field of view (2.3 × 2.3 mm, containing >103 cells, per shot). We used the Photopick platform to evolve archaerhodopsin-derived genetically encoded voltage indicators (GEVIs) with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). These GEVIs gave improved signals in cultured neurons and in live mouse brains. By combining targeted in vivo optogenetic stimulation with high-precision voltage imaging, we characterized inhibitory synaptic coupling between individual cortical NDNF (neuron-derived neurotrophic factor) interneurons, and excitatory electrical synapses between individual hippocampal parvalbumin neurons. The QuasAr6 GEVIs are powerful tools for all-optical electrophysiology and the Photopick approach could be adapted to evolve a broad range of biosensors.

Nanoscale Heat Transfer from Magnetic Nanoparticles and Ferritin in an Alternating Magnetic Field.

Recent suggestions of nanoscale heat confinement on the surface of synthetic and biogenic magnetic nanoparticles during heating by radio frequency-alternating magnetic fields have generated intense interest because of the potential utility of this phenomenon for noninvasive control of biomolecular and cellular function. However, such confinement would represent a significant departure from the classical heat transfer theory. Here, we report an experimental investigation of nanoscale heat confinement on the surface of several types of iron oxide nanoparticles commonly used in biological research, using an all-optical method devoid of the potential artifacts present in previous studies. By simultaneously measuring the fluorescence of distinct thermochromic dyes attached to the particle surface or dissolved in the surrounding fluid during radio frequency magnetic stimulation, we found no measurable difference between the nanoparticle surface temperature and that of the surrounding fluid for three distinct nanoparticle types. Furthermore, the metalloprotein ferritin produced no temperature increase on the protein surface nor in the surrounding fluid. Experiments mimicking the designs of previous studies revealed potential sources of the artifacts. These findings inform the use of magnetic nanoparticle hyperthermia in engineered cellular and molecular systems.

Ultraparamagnetic Cells Formed through Intracellular Oxidation and Chelation of Paramagnetic Iron.

Making cells magnetic is a long-standing goal of chemical biology, aiming to enable the separation of cells from complex biological samples and their visualization in vivo using magnetic resonance imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have been largely unsuccessful due to the stringent required chemical conditions. Here, we introduce an alternative approach to making cells magnetic, focused on biochemically maximizing cellular paramagnetism. We show that a novel genetic construct combining the functions of ferroxidation and iron chelation enables engineered bacterial cells to accumulate iron in "ultraparamagnetic" macromolecular complexes, allowing these cells to be trapped with magnetic fields and imaged with MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, nuclear magnetic resonance, biochemical assays, and computational modeling to elucidate the unique mechanisms and capabilities of this paramagnetic concept.

Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function.

Most cellular phenomena of interest to mammalian biology occur within the context of living tissues and organisms. However, today's most advanced tools for observing and manipulating cellular function, based on fluorescent or light-controlled proteins, work best in cultured cells, transparent model species, or small, surgically accessed anatomical regions. Their reach into deep tissues and larger animals is limited by photon scattering. To overcome this limitation, we must design biochemical tools that interface with more penetrant forms of energy. For example, sound waves and magnetic fields easily permeate most biological tissues, allowing the formation of images and delivery of energy for actuation. These capabilities are widely used in clinical techniques such as diagnostic ultrasound, magnetic resonance imaging, focused ultrasound ablation, and magnetic particle hyperthermia. Each of these modalities offers spatial and temporal precision that could be used to study a multitude of cellular processes in vivo. However, connecting these techniques to cellular functions such as gene expression, proliferation, migration, and signaling requires the development of new biochemical tools that can interact with sound waves and magnetic fields as optogenetic tools interact with photons. Here, we discuss the exciting challenges this poses for biomolecular engineering and provide examples of recent advances pointing the way to greater depth in in vivo cell biology.

Photophysics-informed two-photon voltage imaging using FRET-opsin voltage indicators.

Microbial rhodopsin-derived genetically encoded voltage indicators (GEVIs) are powerful tools for mapping bioelectrical dynamics in cell culture and in live animals. Förster resonance energy transfer (FRET)-opsin GEVIs use voltage-dependent changes in opsin absorption to modulate the fluorescence of an attached fluorophore, achieving high brightness, speed, and voltage sensitivity. However, the voltage sensitivity of most FRET-opsin GEVIs has been reported to decrease or vanish under two-photon (2P) excitation. Here we investigated the photophysics of the FRET-opsin GEVIs Voltron1 and 2. We found that the voltage sensitivity came from a photocycle intermediate, not from the opsin ground state. The voltage sensitivities of both GEVIs were nonlinear functions of illumination intensity; for Voltron1, the sensitivity reversed sign under low-intensity illumination. Using photocycle-optimized 2P illumination protocols, we demonstrate 2P voltage imaging with Voltron2 in barrel cortex of a live mouse. These results open the door to high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo.

Optical constraints on two-photon voltage imaging.

Significance: Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits and limitations of one-photon (1P) versus two-photon (2P) voltage imaging are not well characterized. Aim: We consider the optical and biophysical constraints particular to 1P and 2P voltage imaging and compare the imaging properties of commonly used GEVIs under 1P and 2P excitation. Approach: We measure the brightness and voltage sensitivity of voltage indicators from commonly used classes under 1P and 2P illumination. We also measure the decrease in fluorescence as a function of depth in the mouse brain. We develop a simple model of the number of measurable cells as a function of reporter properties, imaging parameters, and desired signal-to-noise ratio (SNR). We then discuss how the performance of voltage imaging would be affected by sensor improvements and by recently introduced advanced imaging modalities. Results: Compared with 1P excitation, 2P excitation requires ∼ 10 4 -fold more illumination power per cell to produce similar photon count rates. For voltage imaging with JEDI-2P in the mouse cortex with a target SNR of 10 (spike height to baseline shot noise), a measurement bandwidth of 1 kHz, a thermally limited laser power of 200 mW, and an imaging depth of > 300 µ m , 2P voltage imaging using an 80-MHz source can record from no more than ∼ 12 neurons simultaneously. Conclusions: Due to the stringent photon-count requirements of voltage imaging and the modest voltage sensitivity of existing reporters, 2P voltage imaging in vivo faces a stringent tradeoff between shot noise and tissue photodamage. 2P imaging of hundreds of neurons with high SNR at a depth of > 300 µ m will require either major improvements in 2P GEVIs or qualitatively new approaches to imaging.

Optical constraints on two-photon voltage imaging.

Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized. Here we compare the photophysical and imaging properties of commonly used GEVIs under 1P and 2P excitation. 2P excitation requires ~104-fold more illumination power per cell to produce comparable photon count rates to 1P excitation, driving a stringent tradeoff between shot noise and tissue photodamage.

Mapping the microscale origins of magnetic resonance image contrast with subcellular diamond magnetometry.

Magnetic resonance imaging (MRI) is a widely used biomedical imaging modality that derives much of its contrast from microscale magnetic field patterns in tissues. However, the connection between these patterns and the appearance of macroscale MR images has not been the subject of direct experimental study due to a lack of methods to map microscopic fields in biological samples. Here, we optically probe magnetic fields in mammalian cells and tissues with submicron resolution and nanotesla sensitivity using nitrogen-vacancy diamond magnetometry, and combine these measurements with simulations of nuclear spin precession to predict the corresponding MRI contrast. We demonstrate the utility of this technology in an in vitro model of macrophage iron uptake and histological samples from a mouse model of hepatic iron overload. In addition, we follow magnetic particle endocytosis in live cells. This approach bridges a fundamental gap between an MRI voxel and its microscopic constituents.

Biomolecular MRI reporters: Evolution of new mechanisms.

Magnetic resonance imaging (MRI) is a powerful technique for observing the function of specific cells and molecules inside living organisms. However, compared to optical microscopy, in which fluorescent protein reporters are available to visualize hundreds of cellular functions ranging from gene expression and chemical signaling to biomechanics, to date relatively few such reporters are available for MRI. Efforts to develop MRI-detectable biomolecules have mainly focused on proteins transporting paramagnetic metals for T1 and T2 relaxation enhancement or containing large numbers of exchangeable protons for chemical exchange saturation transfer. While these pioneering developments established several key uses of biomolecular MRI, such as imaging of gene expression and functional biosensing, they also revealed that low molecular sensitivity poses a major challenge for broader adoption in biology and medicine. Recently, new classes of biomolecular reporters have been developed based on alternative contrast mechanisms, including enhancement of spin diffusivity, interactions with hyperpolarized nuclei, and modulation of blood flow. These novel reporters promise to improve sensitivity and enable new forms of multiplexed and functional imaging.

Non-invasive imaging using reporter genes altering cellular water permeability.

Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging.

Optical resonance imaging: An optical analog to MRI with sub-diffraction-limited capabilities.

We propose here optical resonance imaging (ORI), a direct optical analog to magnetic resonance imaging (MRI). The proposed pulse sequence for ORI maps space to time and recovers an image from a heterodyne-detected third-order nonlinear photon echo measurement. As opposed to traditional photon echo measurements, the third pulse in the ORI pulse sequence has significant pulse-front tilt that acts as a temporal gradient. This gradient couples space to time by stimulating the emission of a photon echo signal from different lateral spatial locations of a sample at different times, providing a widefield ultrafast microscopy. We circumvent the diffraction limit of the optics by mapping the lateral spatial coordinate of the sample with the emission time of the signal, which can be measured to high precision using interferometric heterodyne detection. This technique is thus an optical analog of MRI, where magnetic-field gradients are used to localize the spin-echo emission to a point below the diffraction limit of the radio-frequency wave used. We calculate the expected ORI signal using 15 fs pulses and 87° of pulse-front tilt, collected using f/2 optics and find a two-point resolution 275 nm using 800 nm light that satisfies the Rayleigh criterion. We also derive a general equation for resolution in optical resonance imaging that indicates that there is a possibility of superresolution imaging using this technique. The photon echo sequence also enables spectroscopic determination of the input and output energy. The technique thus correlates the input energy with the final position and energy of the exciton.