Absolute bioluminescence imaging at the single-cell level with a light signal at the Attowatt level

Enomoto, Toshiteru; Kubota, Hidehiro; Mori, Kaneo; Shimogawara, Masahiro; Yoshita, Masahiro; Ohmiya, Yoshihiro; Akiyama, Hidefumi

doi:10.2144/btn-2018-0043

Cited by 10 publications

(13 citation statements)

References 12 publications

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“…Although we have not compared the LuminoCell with other commercial devices, it is of note, however, that this luminometer cannot compete with commercial instruments in terms of detection limits. While this simple luminometer has a detection limit around 40 pW/s of light irradiance (TSL237S-LF has irradiance responsivity of 2.3 kHz/(μW/cm 2 )), devices equipped with expensive PMT tubes, or special CCD cameras offer much better sensitivity in attowatts (Enomoto et al, 2018). The relatively low detection limit of light irradiance is, however, estimated for 1 second of integration time.…”

Section: Discussionmentioning

confidence: 99%

LuminoCell: a versatile and affordable luminometer platform for monitoring in-cell luciferase-based reporters

Weissova

Fafílek

Radaszkiewicz

et al. 2022

Preprint

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Luciferase reporter assays represent a simple and sensitive experimental system in cell and molecular biology to study multiple biological processes. However, the application of these assays is often limited by the costs of conventional luminometer instruments and the versatility of their use in different experimental conditions. Therefore, we aimed to develop a small, affordable luminometer allowing continuous measurement of luciferase activity, designed for inclusion into various kinds of tissue culture incubators. Here we introduce LuminoCell - an open-source platform for the construction of an affordable, sensitive, and portable luminometer capable of real-time monitoring in-cell luciferase activity. The LuminoCell costs $40, requires less than 1 hour to assemble, and it is capable of performing real-time sensitive detection of both magnitude and duration of the activity of major signalling pathways in cell cultures, including receptor tyrosine kinases (EGF, FGF), WNT/β-catenin, and NF-κB. Additionally, we show that the LuminoCell is suitable to be used in cytotoxicity assays as well as for monitoring periodic circadian gene expression.

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Section: Discussionmentioning

confidence: 99%

LuminoCell: a versatile and affordable luminometer platform for monitoring in-cell luciferase-based reporters

Weissova

Fafílek

Radaszkiewicz

et al. 2022

Preprint

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“…Total average CEA in one section was approximately 460 ng/tissue (Supplementary Figure 1). Previous research using radioimmunoassay has suggested that tumor tissues contain Benchmark around 145-17,850 ng/mg CEA [7]. A bicinchoninic acid assay of another frozen section found about 0.03 mg total protein, indicating around 15,000 ng/mg CEA.…”

Section: Introductionmentioning

confidence: 93%

“…Using this LED, time-averaged emission intensity could be controlled linearly to cover a wide dynamic range of about four orders of magnitude for several years. This calibrated BLI system revealed that luciferase had an average light signal at one attowatt, corresponding to a few thousand photons per second per cell [7]. Based on luciferase quantum yield, we could estimate the amount of luciferase protein in a living cell.…”

Section: Introductionmentioning

confidence: 99%

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Quantitative Immunohistochemistry Using an Antibody-Fused Bioluminescent Protein

Wang

Shimajiri

et al. 2020

BioTechniques

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We established a quantitative detection method for immunohistochemistry based on a reference standard light-emitting diode, protein microarray and antibody-fused bioluminescent protein. In this procedure, we calibrated the bioluminescence imaging system and prepared the calibration curve between antigen and antibody-fused bioluminescent protein using a protein microarray. Then we converted the detecting light signal to antigen count via absolute photon number in the bioluminescent images; there was a resulting threefold difference in the target antigen number between normal and cancerous tissues. Our technique can easily compare immunohistological images and evaluate tumor progression in quantitative pathological diagnosis.

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“…Recently, the bioluminescence intensity emitted by a single mammalian cell-expressing beetle luciferase was estimated to be between 1100–2500 photons s −1 , and the number of luciferase molecules expressed inside the cell was estimated to be approximately 2000–3000 molecules [ 97 ]. However, one limiting factor for attaining high intensity could be oxygen availability, especially in deep tissues under anoxic conditions.…”

Section: Is It Possible To Report In Two Dimensions?mentioning

confidence: 99%

Bioluminescence Color-Tuning Firefly Luciferases: Engineering and Prospects for Real-Time Intracellular pH Imaging and Heavy Metal Biosensing

2022

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Firefly luciferases catalyze the efficient production of yellow-green light under normal physiological conditions, having been extensively used for bioanalytical purposes for over 5 decades. Under acidic conditions, high temperatures and the presence of heavy metals, they produce red light, a property that is called pH-sensitivity or pH-dependency. Despite the demand for physiological intracellular biosensors for pH and heavy metals, firefly luciferase pH and metal sensitivities were considered drawbacks in analytical assays. We first demonstrated that firefly luciferases and their pH and metal sensitivities can be harnessed to estimate intracellular pH variations and toxic metal concentrations through ratiometric analysis. Using Macrolampis sp2 firefly luciferase, the intracellular pH could be ratiometrically estimated in bacteria and then in mammalian cells. The luciferases of Macrolampis sp2 and Cratomorphus distinctus fireflies were also harnessed to ratiometrically estimate zinc, mercury and other toxic metal concentrations in the micromolar range. The temperature was also ratiometrically estimated using firefly luciferases. The identification and engineering of metal-binding sites have allowed the development of novel luciferases that are more specific to certain metals. The luciferase of the Amydetes viviani firefly was selected for its special sensitivity to cadmium and mercury, and for its stability at higher temperatures. These color-tuning luciferases can potentially be used with smartphones for hands-on field analysis of water contamination and biochemistry teaching assays. Thus, firefly luciferases are novel color-tuning sensors for intracellular pH and toxic metals. Furthermore, a single luciferase gene is potentially useful as a dual bioluminescent reporter to simultaneously report intracellular ATP and/or luciferase concentrations luminometrically, and pH or metal concentrations ratiometrically, providing a useful tool for real-time imaging of intracellular dynamics and stress.

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Absolute bioluminescence imaging at the single-cell level with a light signal at the Attowatt level

Cited by 10 publications

References 12 publications

LuminoCell: a versatile and affordable luminometer platform for monitoring in-cell luciferase-based reporters

LuminoCell: a versatile and affordable luminometer platform for monitoring in-cell luciferase-based reporters

Quantitative Immunohistochemistry Using an Antibody-Fused Bioluminescent Protein

Bioluminescence Color-Tuning Firefly Luciferases: Engineering and Prospects for Real-Time Intracellular pH Imaging and Heavy Metal Biosensing

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