Selective-plane-activation structured illumination microscopy.

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids.

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.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.

Calcium-induced upregulation of energy metabolism heats neurons during neural activity.

Cellular temperature affects every biochemical reaction, underscoring its critical role in cellular functions. In neurons, temperature not only modulates neurotransmission but is also a key determinant of neurodegenerative diseases. Considering that the brain consumes a disproportionately high amount of energy relative to its weight, neural circuits likely generate a lot of heat, which can increase cytosolic temperature. However, the changes in temperature within neurons and the mechanisms of heat generation during neural excitation remain unclear. In this study, we achieved simultaneous imaging of Ca2+ and temperature using the genetically encoded indicators, B-GECO and B-gTEMP. We then compared the spatiotemporal distributions of Ca2+ responses and temperature. Following neural excitation induced by veratridine, an activator of the voltage-gated Na+ channel, we observed an approximately 2 °C increase in cytosolic temperature occurring 30 s after the Ca2+ response. The temperature elevation was observed in the non-nuclear region, while Ca2+ increased throughout the cell body. Moreover, this temperature increase was suppressed under Ca2+-free conditions and by inhibitors of ATP synthesis. These results indicate that Ca2+-induced upregulation of energy metabolism serves as the heat source during neural excitation.

Protocol to visualize trans-interaction of clustered protocadherin using cIPAD, a fluorescent indicator, in cultured human cells and mouse neurons.

cIPAD is a fluorescent indicator that allows the visualization of trans-interactions of clustered protocadherin (Pcdh), a cell adhesion molecule that mediates neuronal self-recognition. We describe steps for using HEK293T cells to visualize Pcdh trans-interactions across cells as a preliminary experiment before using dissociated mouse neurons. We then detail procedures for visualizing Pcdh trans-interactions between processes originating from the same neurons, which are considered as Pcdh-mediated neuronal self-recognition. For complete details on the use and execution of this protocol, please refer to Kanadome et al.1.

Mitochondrial temperature homeostasis resists external metabolic stresses.

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

Joule heating involving ion currents through channel proteins.

Ion currents associated with channel proteins in the presence of membrane potential are ubiquitous in cellular and organelle membranes. When an ion current occurs through a channel protein, Joule heating should occur. However, this Joule heating seems to have been largely overlooked in biology. Here we show theoretical investigation of Joule heating involving channel proteins in biological processes. We used electrochemical potential to derive the Joule's law for an ion current through an ion transport protein in the presence of membrane potential, and we suggest that heat production and absorption can occur. Simulation of temperature distribution around a single channel protein with the Joule heating revealed that the temperature increase was as small as <10-3 K, although an ensemble of channel proteins was suggested to exhibit a noticeable temperature increase. Thereby, we theoretically investigated the Joule heating of systems containing ensembles of channel proteins. Nerve is known to undergo rapid heat production followed by heat absorption during the action potential, and our simulation of Joule heating for a squid giant axon combined with the Hodgkin-Huxley model successfully reproduced the feature of the heat. Furthermore, we extended the theory of Joule heating to uncoupling protein 1 (UCP1), a solute carrier family transporter, which is important to the non-shivering thermogenesis in brown adipose tissue mitochondria (BATM). Our calculations showed that the Joule heat involving UCP1 was comparable to the literature calorimetry data of BATM. Joule heating of ion transport proteins is likely to be one of important mechanisms of cellular thermogenesis.

Activity-dependent organization of prefrontal hub-networks for associative learning and signal transformation.

Associative learning is crucial for adapting to environmental changes. Interactions among neuronal populations involving the dorso-medial prefrontal cortex (dmPFC) are proposed to regulate associative learning, but how these neuronal populations store and process information about the association remains unclear. Here we developed a pipeline for longitudinal two-photon imaging and computational dissection of neural population activities in male mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC network topology. Using regularized regression methods and graphical modeling, we found that fear conditioning drove dmPFC reorganization to generate a neuronal ensemble encoding conditioned responses (CR) characterized by enhanced internal coactivity, functional connectivity, and association with conditioned stimuli (CS). Importantly, neurons strongly responding to unconditioned stimuli during conditioning subsequently became hubs of this novel associative network for the CS-to-CR transformation. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on the activity-dependent formation of the hub network within the dmPFC.

Visualization of trans homophilic interaction of clustered protocadherin in neurons.

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.

Visualization of trans-interactions of a protocadherin-α between processes originating from single neurons.

Clustered protocadherin (Pcdh), a cell adhesion protein, is involved in the self-recognition and non-self-discrimination of neurons by conferring diversity on the cell surface. Although the roles of Pcdh in neurons have been elucidated, it has been challenging to visualize its adhesion activity in neurons, which is a molecular function of Pcdh. Here, we present fluorescent indicators, named IPADs, which visualize the interaction of protocadherin-α4 isoform (α4). IPADs successfully visualize not only homophilic α4 trans-interactions, but also combinatorial homophilic interactions between cells. The reversible nature of IPADs overcomes a drawback of the split-GFP technique and allows for monitoring the dissociation of α4 trans-interactions. Specially designed IPADs for self-recognition are able to monitor the formation and disruption of α4 trans-interactions between processes originating from the same neurons. We expect that IPADs will be useful tools for obtaining spatiotemporal information on Pcdh interactions in neuronal self-recognition and non-self-discrimination processes.

Insertion of circularly permuted cyan fluorescent protein into the ligand-binding domain of inositol 1,4,5-trisphosphate receptor for enhanced FRET upon binding of fluorescent ligand.

Binding of fluorescent ligand (FL) to the cyan fluorescent protein (CFP)-coupled ligand-binding domain of the inositol 1,4,5-trisphosphate (IP3) receptor (CFP-LBP) produces fluorescence (Förster) resonance energy transfer (FRET). A competitive fluorescent ligand assay (CFLA), using the FRET signal from competition between FLs and IP3, can measure IP3 concentration. The FRET signal should be enhanced by attaching a FRET donor to an appropriate position. Herein, we inserted five different circularly permuted CFPs in the loop between the second and third α-helices to generate membrane-targeted fluorescent ligand-binding proteins (LBPs). Two such proteins, LBP-cpC157 and LBP-cpC173, localized at the plasma membrane, displayed FRET upon binding the high-affinity ligand fluorescent adenophostin A (F-ADA), and exhibited a decreased fluorescence emission ratio (480 nm / 535 nm) by 1.6- to 1.8-fold that of CFP-LBP. In addition, binding of a fluorescent low-affinity ligand (F-LL) also reduced the fluorescence ratio in a concentration-dependent manner, with EC50 values for LBP-cpC157 and LBP-cpC173 of 34.7 nM and 27.6 nM, respectively. These values are comparable to that with CFP-LBP (29.2 nM), indicating that insertion of cpC157 and cpC173 did not disrupt LBP structure and function. The effect of 100 nM F-LL on the decrease in fluorescence ratio was reversed upon addition of IP3, indicating binding competition between F-LL and IP3. We also constructed cytoplasmic fluorescent proteins cyLBP-cpC157 and cyLBP-cpC173, and bound them to DYK beads for imaging analyses. Application of F-ADA decreased the fluorescence ratio of the beads from the periphery to the center over 3 - 5 min. Application of F-LL also decreased the fluorescence ratio of cyLBP-cpC157 and cyLBP-cpC173 by 20-25%, and subsequent addition of IP3 recovered the fluorescence ratio in a concentration-dependent manner. The EC50 value and Hill coefficient obtained by curve fitting against the IP3-dependent recovery of fluorescence ratio can be used to estimate the IP3 concentration.

Extension of the short wavelength side of fluorescent proteins using hydrated chromophores, and its application.

To perform correlation analysis between different physiological parameters using fluorescent protein-based functional probes, diversification of wavelength properties of fluorescent proteins is underway. However, the shortest emission wavelength of fluorescent proteins has not been updated for more than 10 years. Here, we report the development of Sumire, a fluorescent protein emitting 414 nm violet fluorescence from a hydrated chromophore. The Sumire's fluorescence property allows for the creation of FRET probes that can be used simultaneously with CFP-YFP based FRET probes for multi-parameter analysis.

Development of intensiometric indicators for visualizing N-cadherin interaction across cells.

N-cadherin (NCad) is a classical cadherin that mediates cell-cell interactions in a Ca2+-dependent manner. NCad participates in various biological processes, from ontogenesis to higher brain functions, though the visualization of NCad interactions in living cells remains limited. Here, we present intensiometric NCad interaction indicators, named INCIDERs, that utilize dimerization-dependent fluorescent proteins. INCIDERs successfully visualize reversible NCad interactions across cells. Compared to FRET-based indicators, INCIDERs have a ~70-fold higher signal contrast, enabling clear identification of NCad interactions. In primary neuronal cells, NCad interactions are visualized between closely apposed processes. Furthermore, visualization of NCad interaction at cell adhesion sites in dense cell populations is achieved by two-photon microscopy. INCIDERs are useful tools in the spatiotemporal investigation of NCad interactions across cells; future research should evaluate the potential of INCIDERs in mapping complex three-dimensional architectures in multi-cellular systems.

Fluorescence Imaging of Extracellular Potassium Ion Using Potassium Sensing Oligonucleotide.

Potassium-sensing oligonucleotide, PSO, a conjugate of a quadruplex structure-forming oligonucleotide with a peptide incorporating a Förster Resonance Energy Transfer (FRET) chromophore pair, has been developed for fluorescent detection of potassium ion (K+) in aqueous medium. PSO 1 could be introduced into cells for real-time imaging of cytoplasmic K+ concentrations. To perform fluorescent imaging of K+ on the cell surface, we synthesized twelve PSO derivatives with different types of peptide types and lengths, and oligonucleotide sequences including thrombin-binding aptamer (TBA) sequences with FAM and TAMRA as a FRET chromophore pair, and evaluated their performance. 1 was shown to respond selectively to K+, not to most ions present in vivo, and to show reciprocal fluorescence changes in response to K+ concentration. For the peptide chains and oligonucleotide sequences examined in this study, the PSO derivatives had K d values for K+ in the range of 5-30 mM. All PSO derivatives showed high K+ selectivity even in the presence of excess Na+. The PSO derivatives were successfully localized to the cell surface by biotinylated concanavalin A (ConA) or sulfo-NHS-biotin via streptavidin (StAv). Fluorescence imaging of extracellular K+ upon addition of apoptosis inducers was successfully achieved by 1 localized to the cell surface.

Application of Green-enhanced Nano-lantern as a bioluminescent ratiometric indicator for measurement of Arabidopsis thaliana root apoplastic fluid pH.

Plant root absorbs water and nutrients from the soil, and the root apoplastic fluid (AF) is an important intermediate between cells and the surrounding environment. The acid growth theory suggests that an acidic AF is needed for cell wall expansion during root growth. However, technical limitations have precluded the quantification of root apoplastic fluid pH (AF-pH). Here, we used Green-enhanced Nano-lantern (GeNL), a chimeric protein of the luciferase NanoLuc (Nluc) and the green fluorescent protein mNeonGreen (mNG), as a ratiometric pH indicator based on the pH dependency of bioluminescence resonance energy transfer efficiency from Nluc to mNG. Luminescence spectrum of GeNL changed reciprocally from pH 4.5 to 7.5, with a pKa of 5.5. By fusing GeNL to a novel signal peptide from Arabidopsis thaliana Cellulase 1, we localised GeNL in A. thaliana AF. We visualised AF dynamics at subcellular resolution over 30 min and determined flow velocity in the maturation zone to be 0.97± 0.06 µm/s. We confirmed that the developing root AF is acidic in the pH range of 5.1-5.7, suggesting that the AF-pH is tightly regulated during root elongation. These results support the acid growth theory and provide evidence for AF-pH maintenance despite changes in ambient pH.

Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz Temperature Imaging with a Genetically Encoded Nanothermometer.

Despite improved sensitivity of nanothermometers, direct observation of heat transport inside single cells has remained challenging for the lack of high-speed temperature imaging techniques. Here, we identified insufficient temperature resolution under short signal integration time and slow sensor kinetics as two major bottlenecks. To overcome the limitations, we developed B-gTEMP, a nanothermometer based on the tandem fusion of mNeonGreen and tdTomato fluorescent proteins. We visualized the propagation of heat inside intracellular space by tracking the temporal variation of local temperature at a time resolution of 155 µs and a temperature resolution 0.042 °C. By comparing the fast in situ temperature dynamics with computer-simulated heat diffusion, we estimated the thermal diffusivity of live HeLa cells. The present thermal diffusivity in cells was about 1/5.3 of that of water and much smaller than the values reported for bulk tissues, which may account for observations of heterogeneous intracellular temperature distributions.

Method for Measuring Bioactive Molecules in Blood by a Smartphone Using Bioluminescent Ratiometric Indicators.

Bioluminescent indicators facilitate determination of bioactive molecules in blood samples with high sensitivity. Using a bright luciferase, its bioluminescence (BL) can be easily detected by conventional light sensing devices. In this chapter, we describe a protocol to measure bioactive molecules in blood by taking the BL images with a smartphone camera. We exemplify the measurement of unconjugated bilirubin (UCBR) concentration in the blood of mice using a ratiometric bioluminescent UCBR indicator, BABI (bilirubin assessment with a bioluminescent indicator), and a smartphone camera. We show the UCBR concentration is easily determined through measuring the variance in the BL color with a smartphone camera. This method provides a practical method to lead to future point-of-care diagnosis with quick and simple procedures.

Structure-based analysis and evolution of a monomerized red-colored chromoprotein from the Olindias formosa jellyfish.

GFP-like chromoproteins (CPs) with non-fluorescence ability have been used as bioimaging probes. Existing CPs have voids in the optical absorption window which limits their extensibility. The development of new CP color is therefore ongoing. Here, we cloned CPs from the jellyfish, Olindias formosa, and developed a completely non-fluorescent monomeric red CP, R-Velour, with an absorption peak at 528 nm. To analyze the photophysical properties from a structural aspect, we determined the crystal structure of R-Velour at a 2.1 Å resolution. R-Velour has a trans-chromophore similar to the green fluorescence protein, Gamillus, derived from the same jellyfish. However, in contrast to the two coplanar chromophoric rings in Gamillus, R-Velour has a large torsion inducing non-fluorescence property. Through site-directed mutagenesis, we surveyed residues surrounding the chromophore and found a key residue, Ser155, which contributes to the generation of four-color variants with the bathochromic and hypsochromic shift of the absorption peak, ranging from 506 to 554 nm. The recently proposed spectrum shift theory, based on the Marcus-Hush model, supports the spectrum shift of these mutants. These findings may support further development of R-Velour variants with useful absorption characteristics for bioimaging, including fluorescence lifetime imaging and photoacoustic imaging.

Live Imaging of cAMP Signaling in D. discoideum Based on a Bioluminescent Indicator, Nano-lantern (cAMP).

Bioluminescence imaging of cellular function is a promising strategy. It has advantages over fluorescence imaging such as high sensitivity, no phototoxicity or no autofluorescence, and compatibility to deep-tissue imaging or optogenetics. However, functional imaging of cellular signaling by bioluminescence is not so easy due to the limited availability of bright bioluminescent indicators.Here we describe a detailed strategy to detect cellular cAMP dynamics by using Nano-lantern (cAMP1.6), one of the brightest bioluminescent indicator for cAMP . Both induced and spontaneous cAMP signaling in social amoeba, with a large and small signal change, respectively, were imaged by this method.