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•The color palette of bright luminescent proteins harnessing FRET is introduced.•The most recent near-infrared emitting luciferins are introduced.•Ca2+, ATP, membrane voltage, and ...Zn2+ chemiluminescent indicators are introduced.•Luciferin-based functional indicators for nitric oxide and highly ROS are introduced.
Nowadays, the application of chemiluminescence imaging is no longer limited to the monitoring of slow biological phenomena such as circadian rhythms and in vivo tracking of cells. The expanded color palette of bright chemiluminescent protein and near-infrared-emitting luciferins have greatly improved the sensitivity of in vivo tracking of cells as well as enabling real-time cellular chemiluminescence imaging with high spatial resolution. Furthermore, functional chemiluminescent indicators based on the novel concept have been developed, providing more information related to physiological and pathological processes in living organisms. In this short review, we summarized the recent protein- and substrate-based approaches for tuning the emission wavelength, as well as for development of the functional indicators.
The interior lumen of acidic organelles (e.g., endosomes, secretory granules, lysosomes and plant vacuoles) is an important platform for modification, transport and degradation of biomolecules as ...well as signal transduction, which remains challenging to investigate using conventional fluorescent proteins (FPs). Due to the highly acidic luminal environment (pH ~ 4.5⁻6.0), most FPs and related sensors are apt to lose their fluorescence. To address the need to image in acidic environments, several research groups have developed acid-tolerant FPs in a wide color range. Furthermore, the engineering of pH insensitive sensors, and their concomitant use with pH sensitive sensors for the purpose of pH-calibration has enabled characterization of the role of luminal ions. In this short review, we summarize the recent development of acid-tolerant FPs and related functional sensors and discuss the future prospects for this field.
Genetically encoded calcium ion (Ca2+) indicators have become very useful and widely used tools for Ca2+ imaging, not only in cellular models, but also in living organisms. However, the in vivo and ...in situ characterization of these indicators is tedious and time consuming, and it does not provide information regarding the suitability of an indicator for particular experimental environments. Thus, initial in vitro evaluation of these tools is typically performed to determine their properties. In this review, we examined the properties of dynamic range, affinity, selectivity, and kinetics for Ca2+ indicators. Commonly used strategies for evaluating these properties are presented. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
► In vivo evaluation of genetically encoded Ca2+ indicators (GECIs) is an important but time-taking procedure. ► Usually, a preliminary in vitro evaluation is performed, to determine their properties. ► These properties of GECIs, dynamic range, affinity, selectivity, kinetics, are described.
Development of fluorescence imaging technology using fluorescent proteins has made it possible to visualize physiological functions in live sample. Nanoscopic technologies such as super-resolution ...imaging have also significantly progressed in recent years. However, since the irradiation of excitation light is inevitable in the fluorescence observation, problems such as phototoxicity and autofluorescence are unavoidable. On the other hand, bioluminescence imaging using luciferase is attracting attention. Bioluminescence, unlike fluorescence, does not require external light illumination, so auto-fluorescence and phototoxicity cannot cause. This advantage of bioluminescence over fluorescence has been recognized for some time, but because the emission signal is weak, it requires long exposure times of tens of minutes and has not been used for live imaging. Recently, however, bright bioluminescent proteins such as NanoLuc, Nano-lantern, and Akaluc have been developed, and live imaging by the bioluminescence has begun to be realized. In addition, some bioluminescent systems have been able to biosynthesize luminescent substrates, producing fully spontaneous animals and plants. In this lecture, I will introduce recent progress on technology development related to fluorescence and bioluminescence imaging and discuss future prospects.
The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in ...various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy PALM) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ∼160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.
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•We visualized chromatin structures and their dynamics in live mammalian cells•Nucleosomes form compact chromatin domains in live cells and move coherently•The domains are organized by nucleosome-nucleosome interactions and cohesin•The domains exist during mitosis and act as building blocks of chromosomes
How a genome is organized and behaves in live cells remains unclear. Nozaki et al. visualized little bunches of chromatin, “chromatin domains,” and their dynamic behavior in live mammalian cells. The domains can work as “Lego blocks” of chromosomes to maintain genetic information throughout the cell cycle.
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.
Thermometers for monitoring cellular temperature Nakano, Masahiro; Nagai, Takeharu
Journal of photochemistry and photobiology. C, Photochemistry reviews,
March 2017, 2017-03-00, 20170301, Letnik:
30
Journal Article
Recenzirano
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•Various kinds of thermometers for monitoring cellular temperature have been developed.•Some thermometers showed heterogeneous temperature distribution and thermogenesis at the ...single-cell level.•There is a discussion on the reliability of fluorescence-based thermometric methods.
Temperature is a critical parameter that influences various biological events and reactions in homeotherms (including mammals), poikilotherms, and plants. However, the spatiotemporal patterns of temperature at the single-cell level remain largely unknown. Various kinds of thermometers have been developed for monitoring temperature at the single-cell level. We introduce some cellular thermometers based on Europium (III) thenoyltrifluoroacetonate trihydrate, gold nanoclusters, nanodiamonds, synthetic polymers, quantum dots, green fluorescent protein, and bimaterial microcantilevers. We also discuss a critique of these thermometric methods and provide some responses to this critique.
Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging ...is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins. eNLs allow five-colour live-cell imaging, as well as detection of single protein complexes and even single molecules. We also develop an eNL-based Ca
indicator with a 500% signal change, which can image spontaneous Ca
dynamics in cardiomyocyte and neural cell models. These eNL probes facilitate not only multicolour imaging in living cells but also sensitive imaging of a wide repertoire of proteins, even at very low expression levels.
Temperature is a fundamental physical parameter that plays an important role in biological reactions and events. Although thermometers developed previously have been used to investigate several ...important phenomena, such as heterogeneous temperature distribution in a single living cell and heat generation in mitochondria, the development of a thermometer with a sensitivity over a wide temperature range and rapid response is still desired to quantify temperature change in not only homeotherms but also poikilotherms from the cellular level to in vivo. To overcome the weaknesses of the conventional thermometers, such as a limitation of applicable species and a low temporal resolution, owing to the narrow temperature range of sensitivity and the thermometry method, respectively, we developed a genetically encoded ratiometric fluorescent temperature indicator, gTEMP, by using two fluorescent proteins with different temperature sensitivities. Our thermometric method enabled a fast tracking of the temperature change with a time resolution of 50 ms. We used this method to observe the spatiotemporal temperature change between the cytoplasm and nucleus in cells, and quantified thermogenesis from the mitochondria matrix in a single living cell after stimulation with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which was an uncoupler of oxidative phosphorylation. Moreover, exploiting the wide temperature range of sensitivity from 5°C to 50°C of gTEMP, we monitored the temperature in a living medaka embryo for 15 hours and showed the feasibility of in vivo thermometry in various living species.
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