Hidehiro Kubota

Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate

Reporter assays that use luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly luciferase and Renilla luciferase are used for monitoring single gene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because one of the two reporting luciferases must be used as an internal control. We have developed a novel reporter assay system in which three luciferases that emit green, orange, and red light with a single substrate are used as reporter genes. The activities of the luciferases can be measured simultaneously and quantitatively with optical filters. This system enables us to simply and rapidly monitor multiple gene expressions in a one-step reaction.

Development of a Quantitative Bio/Chemiluminescence Spectrometer Determining Quantum Yields: Re-examination of the Aqueous Luminol Chemiluminescence Standard

We have developed a bio/chemiluminescence spectrometer with a cooled charge‐coupled‐device (CCD) detector to obtain a quantitative luminescence spectrum as the absolute number of all emitted photons at each wavelength. The integrated area of the spectrum divided by the number of reacted substrate molecules gives the quantum yield. Calibration of the absolute sensitivity of the CCD‐spectrometer system was performed by using lasers and a tungsten lamp with calibrated powers as primary light standards, and calibration of the light‐collection efficiency of the spectrometer with several kinds of cells for liquid samples was achieved by introducing a simple reference double‐plate cell. The reference cell is not convenient for final bio/chemiluminescence measurements but is useful for the calibration because it has well‐defined angular dependence of light emission, allowing accurate calculation of the light‐collection efficiency. Using this CCD‐spectrometer system, we re‐examined the quantum yield of aqueous luminol chemiluminescence with H2O2 catalyzed by horseradish peroxidase. The quantum yield was constant for a wide range of luminol concentrations, whereas it changed and had an optimum against H2O2 concentrations. The optimum quantum yield was 1.23(±0.20)%, which is in good agreement with previously reported values.

Quantum Yields and Quantitative Spectra of Firefly Bioluminescence with Various Bivalent Metal Ions

We measured quantitative spectra of firefly (Photinus pyralis) bioluminescence in the presence of Zn2+ and other bivalent metal ions to investigate the effects of these metal ions on luciferin‐luciferase reaction. We studied the dependence of the quantum yield and spectrum on quantity and kind of bivalent metal ions. Adding various amounts of Mg2+, Mn2+ and Ca2+ produced virtually no change in the quantum yields or the spectra of bioluminescence. In contrast, increasing amounts of ions such as Zn2+ and Cd2+ decreased quantum yields and changed the bioluminescence color from yellow‐green to red. Quantitative analysis showed that the sensitivities of the quantum yield and color to various metal ions were in the order of Hg2+>Zn2+, Cd2+>Ni2+, Co2+, Fe2+≫Mg2+, Mn2+, Ca2+. We propose that the changes in quantum yield and spectrum caused by the metal ions are due to their effect on luciferase that surrounds oxyluciferin during its radioactive decay. We also found that having more metal ions accelerated bioluminescence reactions. The sensitivity of the reaction rate had no correlation with those of the quantum yield and spectrum.

Absolute electroluminescence imaging of multi-junction solar cells and calibration standards

We developed absolute electroluminescence (EL) calibration standards to evaluate absolute radiative-emission rates from subcells in multi-junction (MJ) solar cells. The absolute-EL-measurement system consists of an EL imaging setup and an emission-intensity-calibrated planar light-emitting diode with a circular open aperture as an emission-intensity standard. We applied this system to the measurements of the absolute EL imaging of a monolithic satellite-use InGaP/GaAs/Ge MJ solar cell. From the observed absolute EL images, we characterized external EL quantum efficiencies and internal open-circuit voltages of InGaP and GaAs subcells.

Calibration standards and measurement accuracy of absolute electroluminescence and internal properties in multi-junction and arrayed solar cells

We developed methodologies and calibration standards for absolute electroluminescence (EL) measurements for CONTACT-LESS evaluation of various internal properties of multi-junction and arrayed solar cells, such as open-circuit voltages, external and internal radiative efficiencies, and luminescence-coupling efficiency. Several independent calibration methods were compared that used: 1) a calibrated EL imaging system, 2) proximity measurement with a large-area photodiode, 3) an integrating-sphere system, and 4) planar light-emitting diodes with a circular aperture. The comparison clarified the advantages and disadvantages of each method, and showed consistency within 30% uncertainty, resulting in a 7-meV uncertainty in open-circuit voltage measurements.

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

Bioluminescence imaging (BLI) demonstrates cellular events as a light signal at the single-cell level using a highly sensitive, cooled CCD camera. However, BLI signals are relative values and thus, images taken on different days or using different equipment cannot be compared directly. We established a reference LED light source that was characteristic of the total flux and light distribution and calibrated the BLI system as an absolute light signal. This calibrated BLI system revealed that the average light signal of beetle luciferase was at an attowatt level per sec at the single cell level.

Light-emitting-diode Lambertian light sources as low-radiant-flux standards applicable to quantitative luminescence-intensity imaging

Planar-type Lambertian light-emitting diodes (LEDs) with a circular aperture of several tens of μm to a few mm in diameter were developed for use as radiant-flux standard light sources, which have been in strong demand for applications such as quantitative or absolute intensity measurements of weak luminescence from solid-state materials and devices. Via pulse-width modulation, time-averaged emission intensity of the LED devices was controlled linearly to cover a wide dynamic range of about nine orders of magnitude, from 10 μW down to 10 fW. The developed planar LED devices were applied as the radiant-flux standards to quantitative measurements and analyses of photoluminescence (PL) intensity and PL quantum efficiency of a GaAs quantum-well sample. The results demonstrated the utility and applicability of the LED standards in quantitative luminescence-intensity measurements in Lambertian-type low radiant-flux level sources.

Accuracy evaluations for standardization of multi-junction solar-cell characterizations via absolute electroluminescence

Absolute electroluminescence (EL) measurements have been becoming essential methods to evaluate internal properties of individual subcells in multi-junction (MJ) solar cells. To establish methodology of absolute EL for standardization of MJ solar-cell characterizations, we developed independently calibrated absolute EL methods and verified accuracy and uncertainties via round-robin studies measuring EL emission rates for InGaP/GaAs/Ge triple-junction and single-junction solar cells. The results showed good quantitative agreements within 10% discrepancy among the individually calibrated methods, which corresponds to less-3-meV uncertainties in the estimation of the open-circuit voltages. This enables accurate comparison of the deviations between the open-circuit voltages via the transport experiment and via the absolute EL methods and detailed characterization of the internal properties of MJ solar cells.