2-photon Fluorescence Lifetime Imaging Research Articles

Probing intracellular potassium dynamics in neurons with the genetically encoded sensor lc-LysM GEPII 1.0 in vitro and in vivo

Neuronal activity is accompanied by a net outflow of potassium ions (K+) from the intra- to the extracellular space. While extracellular [K+] changes during neuronal activity are well characterized, intracellular dynamics have been less well investigated due to lack of respective probes. In the current study we characterized the FRET-based K+ biosensor lc-LysM GEPII 1.0 for its capacity to measure intracellular [K+] changes in primary cultured neurons and in mouse cortical neurons in vivo. We found that lc-LysM GEPII 1.0 can resolve neuronal [K+] decreases in vitro during seizure-like and intense optogenetically evoked activity. [K+] changes during single action potentials could not be recorded. We confirmed these findings in vivo by expressing lc-LysM GEPII 1.0 in mouse cortical neurons and performing 2-photon fluorescence lifetime imaging. We observed an increase in the fluorescence lifetime of lc-LysM GEPII 1.0 during periinfarct depolarizations, which indicates a decrease in intracellular neuronal [K+]. Our findings suggest that lc-LysM GEPII 1.0 can be used to measure large changes in [K+] in neurons in vitro and in vivo but requires optimization to resolve smaller changes as observed during single action potentials.

Cannabidiol Modulates Mitochondrial Redox and Dynamics in MCF7 Cancer Cells: A Study Using Fluorescence Lifetime Imaging Microscopy of NAD(P)H.

The cannabinoid, cannabidiol (CBD), is part of the plant's natural defense system that when given to animals has many useful medicinal properties, including activity against cancer cells, modulation of the immune system, and efficacy in epilepsy. Although there is no consensus on its precise mode of action as it affects many cellular targets, CBD does appear to influence mitochondrial function. This would suggest that there is a cross-kingdom ability to modulate stress resistance systems that enhance homeostasis. As NAD(P)H autofluorescence can be used as both a metabolic sensor and mitochondrial imaging modality, we assessed the potential of this technique to study the in vitro effects of CBD using 2-photon excitation and fluorescence lifetime imaging microscopy (2P-FLIM) of NAD(P)H against more traditional markers of mitochondrial morphology and cellular stress in MCF7 breast cancer cells. 2P-FLIM analysis revealed that the addition of CBD induced a dose-dependent decrease in bound NAD(P)H, with 20 µM treatments significantly decreased the contribution of bound NAD(P)H by 14.6% relative to the control (p < 0.001). CBD also increased mitochondrial concentrations of reactive oxygen species (ROS) (160 ± 53 vs. 97.6 ± 4.8%, 20 µM CBD vs. control, respectively, p < 0.001) and Ca2+ (187 ± 78 vs. 105 ± 10%, 20 µM CBD vs. the control, respectively, p < 0.001); this was associated with a significantly decreased mitochondrial branch length and increased fission. These are all suggestive of mitochondrial stress. Our results support the use of NAD(P)H autofluorescence as an investigative tool and provide further evidence that CBD can modulate mitochondrial function and morphology in a dose-dependent manner, with clear evidence of it inducing oxidative stress at higher concentrations. This continues to support emerging data in the literature and may provide further insight into its overall mode of action, not only in cancer, but potentially its function in the plant and why it can act as a medicine.

Quenched coumarin derivatives as fluorescence lifetime phantoms for NADH and FAD

Two-photon fluorescence lifetime imaging is a versatile laboratory technique in the field of biophotonics and its importance is also growing in the field of in vivo diagnostics for medical purposes. After years of experience in dermatology, endoscopic implementations of the technique are now posing new technical challenges. To develop, test, and compare instrumental solutions for this purpose suitable reference samples have been devised and tested. These reference samples can serve as reliable NADH- and FAD-mimicking optical phantoms for 2-photon fluorescence lifetime imaging, as they can be prepared relatively easily with reproducible and stable characteristics for this quite relevant diagnostic technique. The reference samples (mixtures of coumarin 1 and coumarin 6 in ethanol with suitable amounts of 4-hydroxy-TEMPO) have been tuned to exhibit spectral and temporal fluorescence characteristics very similar to those of NADH and FAD, the two molecules most frequently utilized to characterize cell metabolism.

Delivery of AAV for Expression of Fluorescent Biosensors in Juvenile Mouse Hippocampus.

Genetically encoded fluorescent biosensors are versatile tools for studying brain metabolism and function in live tissue. The genetic information for these biosensors can be delivered into the brain by stereotaxic injection of engineered adeno-associated viruses (AAVs), which can selectively target different cell types depending on the capsid serotype and/or the viral promoter. Here, we describe a protocol for intracranial injections of two viral vectors encoding the metabolic biosensor Peredox and the calcium biosensor RCaMP1h. When combined with 2-photon microscopy and fluorescence lifetime imaging, this protocol allows the simultaneous quantitative assessment of changes in the cytosolic NADH/NAD+ ratio and the intracellular Ca2+ levels in individual dentate granule cells from acute hippocampal slices. Graphic abstract: Workflow diagram for biosensor expression in the mouse hippocampus using intracranial injections of adeno-associated viruses.

Two-Photon Phosphorescence Lifetime Microscopy.

Two-photon Phosphorescence Lifetime Microscopy (2PLM) is an emerging nonlinear optical technique that has great potential to improve our understanding of the basic biology underlying human health and disease. Although analogous to 2-photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), the contrast in 2PLM is fundamentally different from various intensity-based forms of imaging since it is based on the lifetime of an excited state and can be regarded as a "functional imaging" technique. 2PLM signal originates from the deactivation of the excited triplet state (phosphorescence) [1, 2]. Typically, this triplet state is a much longer-lived excited state than the singlet excited state resulting in phosphorescence emission times of microseconds to milliseconds at room temperature as opposed to nanoseconds for fluorescence emission [3]. The long-lived nature of the triplet state makes it highly sensitive to quenching molecules in the surrounding environment such as biomolecular oxygen (O2). Therefore, 2PLM can provide not only information on the distribution pattern of the probe in the sample (via intensity) but also determine the local oxygen tension (via phosphorescence lifetime quenching) [1]. The ability to create three-dimensional optical sections in the plane of focus within a thick biological specimen while maintaining relatively low phototoxicity due to the use of near-infrared wavelengths for two-photon excitation gives 2PLM powerful advantages over other techniques for longitudinal imaging and monitoring of oxygen within living organisms [4]. In this chapter, we will provide background on the development of 2PLM, discuss the most common oxygen sensing measurement methods and concepts, and explain the general principles and optical configuration of a 2PLM system. We also discuss the key characteristics and strategies for improvement of the technique. Finally, we will present an overview of the current primary scientific literature of how 2PLM has been used for oxygen sensing in biological applications and how this technique is improving our understanding of the basic biology underlying several areas of human health.

Calcineurin Inhibition Impairs the Function of Neuronal Potassium‐Chloride Cotransporter 2

Calcineurin inhibitors (CnI) such as Cyclosporin A (CsA) are instrumental for immunosuppression after organ transplantation but may cause serious neurologic side effects, including seizures.Neuronal excitability depends on intact Cl− homeostasis. Generation of hyperpolarizing synaptic responses to GABA and glycine requires a Cl− gradient across the cell membrane with a low [Cl−]i, mainly established by K+‐Cl− cotransporter 2 (KCC2) and to a lesser extent Na+‐K+‐Clcotransporter 1 (NKCC1); both are cation‐coupled chloride cotransporters (CCCs). Calcineurin has been implicated in the regulation of CCCs, whereas impaired CCC function is a well‐known condition in human pharmacoresistant epilepsy. Therefore, we hypothesized that CsA affects KCC2 or NKCC1 functions, thus causing neuronal hyperexcitability.In ex vivo intracellular recordings with sharp microelectrodes, Wistar rat pyramidal neurons in neocortical slices responded to CsA (5 μM for 1h) with a less negative GABAA reversal potential (+7.2 mV) and prolonged Cl− extrusion time after iontophoretic Cl− loading (+3.9 s). 2‐photon fluorescence lifetime imaging in presence of Cl− sensitive dye MQAE showed an increased [Cl−]i in layer V neurons (+6.9 mM), suggesting reduced KCC2 activity. Co‐immunoprecipitation studies in rodent brain tissue suggested interactions of calcineurin Aβ (CnAβ) with KCC2 and NKCC1.In vivo, CsA administration to rats (5–25 mg/kg i.p.) enhanced levels of inhibitory tyrosine KCC2 phosphorylation at short term (4h; +172%) and reduced levels of activating S940‐KCC2 phosphorylation in the long term (14d; −61%). Reduced phospho‐S940‐KCC2 levels were observed in further models of calcineurin inhibition such as genetic CnAβ‐deficiency in mice (−78%) or CsA treatment of zebrafish larvae (10 μM in water: −54% after 24h). In contrast, NKCC1 and its activating kinase SPAK were stimulated upon calcineurin inhibition in rodents. Similar data were obtained in Drosophila melanogaster.Our results provide evidence that CnI attenuate KCC2 function but may stimulate NKCC1 leading to elevated [Cl−]i. These effects may increase neuronal excitability and contribute to CnI neurological adverse effects. The benefits of KCC2 activators or SPAK inhibitors for enhancing chloride extrusion in patients experiencing CnI neurotoxicity deserve further characterization.Support or Funding InformationThis study was supported by Deutsche Forschergemeinschaft.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vivo Imaging of Cellular Excitation-Transcription Coupling in the Mouse Brain

Regulation of gene transcription is essential for long-term plasticity in the brain. However, it has not been possible to monitor dynamics of neuronal transcription in vivo and to correlate them to neuronal activity patterns at cellular resolution. Here we present a technique to image in vivo signaling of cAMP response element binding protein (CREB), an activity-dependent transcription factor important for synaptic plasticity, using 2-photon fluorescence lifetime imaging (2pFLIM) in combination with new FRET biosensors. This allowed for long-term simultaneous imaging of CREB and neuronal activity in cortical neurons of awake animals. We found that, on average, CREB activity is positively correlated with behavioral performance in a motor learning task. Further, we performed chronic dual-imaging of CREB and neuronal activity in primary visual cortex, identifying a correlation between CREB and neuronal activity dynamics following sensory deprivation. This approach will allow researchers to unravel transcriptional dynamics underlying experience-dependent plasticity in the brain.

Application of label‐free 2‐photon fluorescence lifetime imaging microscopy to measure endogenous melanin profiles in human eye melanocytes, naevus and melanoma cells

PurposeThe progression of choroidal melanoma (CM) is complex, involving genetic and immune‐related factors. Melanin/pigmentation genes and forms (eumelanin/dark and pheomelanin/light) can also impact on CM progression. In this study, an optimised 2‐photon fluorescence lifetime imaging microscopy (FLIM), with phasor analysis, was used to identify melanin fluorescence lifetime (FL) profiles in formalin‐fixed, label‐free paraffin sections of human melanoma and naevus, with surrounding heterogeneously pigmented melanocytes.MethodsSections of ‘light CM’ (n = 3) with ‘mixed surrounding melanocytes’ and ‘dark naevi’ (n = 3) with ‘dark surrounding melanocytes’ were imaged with FLIM (3 sampled regions). Fluorescence lifetimes were measured at every pixel of captured FLIM images, Fourier transformed and presented in a ‘fit‐free’ phasor plot. These plots were segmented by 7 phasor clusters of linearly increasing fluorescence lifetimes mapped to intracellular melanin in melanocytes, naevus and melanoma cells. The mean fraction of FLIM image pixels linked to each melanin‐mapped cluster was obtained for all sampled regions to form melanin FL profiles.ResultsThe measured sampled regions displayed distinct intracellular melanin (eumelanin:pheomelanin) profiles with varying dominant melanin‐mapped clusters. The dominant ‘highest pixel fraction’ cluster measured in ‘light CM’ mapped to long FLs, implying a low eu:pheo ratio. The ‘mixed pigmented melanocytes around CM’ showed a mixed eu:pheo content based on the dominant mid‐valued FL cluster. The ‘dark naevi and surrounding melanocytes’ were mapped to mostly short FLs (high eu:pheo ratio).ConclusionsOur FLIM‐phasor method provides a fast ‘model‐free’ way to unmix melanin fluorescence lifetimes in melanocytes, naevi and CM, and provides a basis for exploring the role of melanin forms in eye melanoma pathogenesis.

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo.

Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics.

Development of a molecularly evolved, highly sensitive CaMKII FRET sensor with improved expression pattern.

Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca2+/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors.

Revealing the Cellular Metabolic State through NADH Autofluorescence Lifetime in Parkinson's Disease

Cellular metabolic imaging of the relative amounts of reduced NADH and the microenvironment of this metabolic electron carrier can be used to noninvasively monitor changes in cell metabolism in various diseases. Moreover, mitochondrial dysfunction, which is directly related to the cellular metabolic state, is considered to be central in developing Parkinson's disease (PD). We applied 2-photon fluorescence lifetime imaging microscopy to determine the fluorescence lifetime as well as the amounts of reduced NADH in PC12 cells (a cell line derived from pheochromocytoma of rat adrenal medulla) treated with Neuronal Growth Factor (NGF) to differentiate into neuronal cells. The neuronal cells were further treated with MPTP to induce PD syndromes and the change in fluorescence lifetime and the amounts of reduced NADH after the treatment were measured. Our preliminary results show a significant increase in the fluorescence lifetime of free as well as bound NADH in the PD induced cells. Moreover, the relative amounts of the ratio of free NADH over bound NADH does not show any significant difference among the control and PD cells. These results may lead to further understanding of the role of cellular metabolism in PD progression.

A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging.

Neuromodulators have profound effects on behavior, but the dynamics of their intracellular effectors has remained unclear. Most neuromodulators exert their function via G-protein-coupled receptors (GPCRs). One major challenge for understanding neuromodulator action is the lack of dynamic readouts of the biochemical signals produced by GPCR activation. The adenylate cyclase/cyclic AMP/protein kinase A (PKA) module is a central component of such biochemical signaling. This module is regulated by several behaviorally important neuromodulator receptors. Furthermore, PKA activity is necessary for the induction of many forms of synaptic plasticity as well as for the formation of long-term memory. In order to monitor PKA activity in brain tissue, we have developed a 2-photon fluorescence lifetime imaging microscopy (2pFLIM) compatible PKA sensor termed FLIM-AKAR, which is based on the ratiometric FRET sensor AKAR3. FLIM-AKAR shows a large dynamic range and little pH sensitivity. In addition, it is a rapidly diffusible cytoplasmic protein that specifically reports net PKA activity in situ. FLIM-AKAR expresses robustly in various brain regions with multiple transfection methods, can be targeted to genetically identified cell types, and responds to activation of both endogenous GPCRs and spatial-temporally specific delivery of glutamate. Initial experiments reveal differential regulation of PKA activity across subcellular compartments in response to neuromodulator inputs. Therefore, the reporter FLIM-AKAR, coupled with 2pFLIM, enables the study of PKA activity in response to neuromodulator inputs in genetically identified neurons in the brain, and sheds light on the intracellular dynamics of endogenous GPCR activation.

An Improved Ras Sensor for Highly Sensitive and Quantitative FRET-FLIM Imaging

Ras is a signaling protein involved in a variety of cellular processes. Hence, studying Ras signaling with high spatiotemporal resolution is crucial to understanding the roles of Ras in many important cellular functions. Previously, fluorescence lifetime imaging (FLIM) of fluorescent resonance energy transfer (FRET)-based Ras activity sensors, FRas and FRas-F, have been demonstrated to be useful for measuring the spatiotemporal dynamics of Ras signaling in subcellular micro-compartments. However the predominantly nuclear localization of the sensors' acceptor has limited its sensitivity. Here, we have overcome this limitation and developed two variants of the existing FRas sensor with different affinities: FRas2-F (Kd∼1.7 µM) and FRas2-M (Kd∼0.5 µM). We demonstrate that, under 2-photon fluorescence lifetime imaging microscopy, FRas2 sensors provide higher sensitivity compared to previous sensors in 293T cells and neurons.

Imaging Rho GTPases Activation in Single Dendritic Spines by 2-photon Fluorescence Lifetime Imaging Microscopy

Imaging Rho GTPases Activation in Single Dendritic Spines by 2-photon Fluorescence Lifetime Imaging Microscopy

ErbB-2 Blockade and Prenyltransferase Inhibition Alter Epidermal Growth Factor and Epidermal Growth Factor Receptor Trafficking and Enhance 111In-DTPA-hEGF Auger Electron Radiation Therapy

The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of (111)In-labeled human epidermal growth factor ((111)In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of (111)In-DTPA-hEGF. The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. (111)In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of (111)In-DTPA-hEGF in MDA-MB-468 (1.3 × 10(6) EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 × 10(6) EGFR per cell; 0.4 × 10(5) ErbB-2 per cell) cell lines. The cytotoxicity of (111)In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 μg/mL) or L-778,123 (0-22.5 μM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with (111)In-DTPA-hEGF plus trastuzumab or L-788,123. Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of (111)In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to (111)In-DTPA-hEGF. Nuclear localization and cytotoxicity of (111)In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P < 0.0001). The therapeutic efficacy of (111)In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.

1SM-06 シナプス内信号伝達、2光子励起蛍光寿命イメージング、神経生物科学"単一シナプスにおける信号伝達の可視化"(1SM 異分野融合する1分子研究:コンフォートゾーンを深めて超えて,日本生物物理学会第49回年会(2011年度))

1SM-06 シナプス内信号伝達、2光子励起蛍光寿命イメージング、神経生物科学"単一シナプスにおける信号伝達の可視化"(1SM 異分野融合する1分子研究:コンフォートゾーンを深めて超えて,日本生物物理学会第49回年会(2011年度))

3K1522 二光子蛍光寿命イメージング法によるRho GTPase活性化の単一シナプスレベル可視化解析(細胞生物的課題4,第49回年会講演予稿集)

3K1522 二光子蛍光寿命イメージング法によるRho GTPase活性化の単一シナプスレベル可視化解析(細胞生物的課題4,第49回年会講演予稿集)

Two-Photon Lifetime Imaging of Signal Transduction in Neurons

The ability of neuron to change and maintain biochemical signaling, shape and the size of synapses in response to stimuli is known as synaptic plasticity, which is believed to be the cellular basis for learning and memory. Recent advance in studying synaptic plasticity is 2-photon Fluorescence Lifetime Imaging Microscopy (2pFLIM), which enables the visualization of the activity and oligomerization of signaling molecules at the level of single synapse in neurons. Here we introduce the recent results of the imaging of CamKII and Ras activation in single dendritic spines in brain tissue.

Spatiotemporal Regulation of Signaling in and out of Dendritic Spines: CaMKII and Ras.

Recent advances in 2-photon fluorescence lifetime imaging microscopy (2pFLIM) in combination with 2-photon photochemistry have enabled the visualization of neuronal signaling during synaptic plasticity at the level of single dendritic spines in light scattering tissue. Using these techniques, the activity of Ca(2+)/Calmodulin-dependent kinase II (CaMKII) and Ras have been imaged in single spines during synaptic plasticity and associated spine enlargement. These provide two contrasting examples of spatiotemporal regulation of spine signaling: Ras signaling is diffusive and spread over ~10 μm along the dendrites, while CaMKII activation is restricted to the spine undergoing plasticity. In this review, we will discuss the mechanisms and roles of the different spatiotemporal regulation of signaling in neurons, and the impact of the spine structure upon these biochemical signaling processes.

1TA5-03 二光子蛍光寿命イメージング法によるRho GTPase活性化の単一シナプスレベル可視化解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

1TA5-03 二光子蛍光寿命イメージング法によるRho GTPase活性化の単一シナプスレベル可視化解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)