Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain.

Blue-shifted genetically encoded Ca indicator with enhanced two-photon absorption.

Significance: Genetically encoded calcium ion () indicators (GECIs) are powerful tools for monitoring intracellular concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited.

High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.

l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (Δ/ = 15 to 30 ), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities.

Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability.

A blue-shifted genetically encoded Ca indicator with enhanced two-photon absorption.

Significance: Genetically encoded calcium ion (Ca) indicators (GECIs) are powerful tools for monitoring intracellular Ca concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited.

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging.

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.

Quantitative assessment of near-infrared fluorescent proteins.

Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins (NIR FPs), which are rapidly becoming popular probes for a variety of imaging applications. However, the diversity of NIR FPs poses a challenge for end-users in choosing the optimal one for a given application. Here we conducted a systematic and quantitative assessment of intracellular brightness, photostability, oligomeric state, chemical stability and cytotoxicity of 22 NIR FPs in cultured mammalian cells and primary mouse neurons and identified a set of top-performing FPs including emiRFP670, miRFP680, miRFP713 and miRFP720, which can cover a majority of imaging applications.

A genetically encoded far-red fluorescent indicator for imaging synaptically released Zn.

Synaptic zinc ion (Zn) has emerged as a key neuromodulator in the brain. However, the lack of research tools for directly tracking synaptic Zn in the brain of awake animals hinders our rigorous understanding of the physiological and pathological roles of synaptic Zn. In this study, we developed a genetically encoded far-red fluorescent indicator for monitoring synaptic Zn dynamics in the nervous system.

A genetically encoded far-red fluorescent calcium ion biosensor derived from a biliverdin-binding protein.

Far-red and near-infrared (NIR) genetically encoded calcium ion (Ca ) indicators (GECIs) are powerful tools for in vivo and multiplexed imaging of neural activity and cell signaling. Inspired by a previous report to engineer a far-red fluorescent protein (FP) from a biliverdin (BV)-binding NIR FP, we have developed a far-red fluorescent GECI, designated iBB-GECO1, from a previously reported NIR GECI. iBB-GECO1 exhibits a relatively high molecular brightness, an inverse response to Ca with ΔF/F  = -13, and a near-optimal dissociation constant (K ) for Ca of 105 nM.

Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It.

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750-800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates at different excitation wavelengths across the whole two-photon absorption spectrum.

Rapid directed molecular evolution of fluorescent proteins in mammalian cells.

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10 independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days.

A genetically encoded fluorescent biosensor for extracellular L-lactate.

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.

Voltage Imaging in Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter.

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in . Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach to achieve selective neuron labeling in intact fly brains.

A fast, high-affinity fluorescent serotonin biosensor engineered from a tick lipocalin.

Serotonin (5-HT) is an important signaling monoamine and neurotransmitter. We report structure-guided engineering of a green fluorescent, genetically encoded serotonin sensor (G-GESS) from a 5-HT-binding lipocalin in the soft tick Argas monolakensis. G-GESS shows fast response kinetics and high affinity, specificity, brightness and photostability.

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease.

Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range.

Two-photon laser scanning microscopy (2PLSM) is a state-of-the-art technique used for non-invasive imaging deep inside the tissue, with high 3D resolution, minimal out-of-focus photodamage, and minimal autofluorescence background. For optimal application of fluorescent probes in 2PLSM, their two-photon absorption (2PA) spectra, expressed in absolute cross sections must be characterized. Excitation at optimum wavelength will make it possible to reduce the laser power and therefore minimize photodamage.

Correction to: A genetically encoded Ca indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

Understanding the Fluorescence Change in Red Genetically Encoded Calcium Ion Indicators.

For over 20 years, genetically encoded Ca indicators have illuminated dynamic Ca signaling activity in living cells and, more recently, whole organisms. We are just now beginning to understand how they work. Various fluorescence colors of these indicators have been developed, including red.

A genetically encoded near-infrared fluorescent calcium ion indicator.

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca imaging in combination with other optogenetic indicators and actuators.