Quantitative Analysis of Bioluminescence Optical Signal.

Bioluminescence is light emission based on the luciferin-luciferase enzymatic reaction in living organisms. Optical signals from bioluminescence (BL) reactions are available for bioanalysis and bioreporters for gene expression, in vitro, in vivo, and ex vivo bioimaging, immunoassay, and other applications. Although there are numerous bioanalysis methods based on BL signal measurements, the BL signal is measured as a relative value, and not as an absolute value. Recently, some approaches have been established to completely quantify the BL signal, resulting in, for instance, the redetermination of the quantum yield of the BL reaction and counting the photon number of the BL signal at the single-cell level. Reliable and reproducible understanding of biological events in the bioanalysis and bioreporter fields can be achieved by means of standardized absolute optical signal measurements, which is described in an International Organization for Standardization (ISO) document.

Quantitative immunohistochemistry using an antibody-fused bioluminescent protein.

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.

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.

Strongly Chemiluminescent Acridinium Esters under Neutral Conditions: Synthesis, Properties, Determination, and Theoretical Study.

Various novel acridinium ester derivatives having phenyl and biphenyl moieties were synthesized, and their optimal chemiluminescence conditions were investigated. Several strongly chemiluminescent acridinium esters under neutral conditions were found, and then these derivatives were used to detect hydrogen peroxide and glucose. Acridinium esters having strong electron-withdrawing groups such as cyano, methoxycarbonyl, and nitro at the 4-position of the phenyl moiety in phenyl 10-methyl-10λ4-acridine-9-carboxylate trifluoromethanesulfonate salt showed strong chemiluminescence intensities. The chemiluminescence intensity of 3,4-dicyanophenyl 10-methyl-10λ4-acridine-9-carboxylate trifluoromethanesulfonate salt was approximately 100 times stronger than that of phenyl 10-methyl-10λ4-acridine-9-carboxylate trifluoromethanesulfonate salt at pH 7. The linear calibration ranges of hydrogen peroxide and glucose were 0.05-10 mM and 10-2000 µM using 3,4-(dimethoxycarbonyl)phenyl 10-methyl-10λ4-acridine-9-carboxylate trifluoromethanesulfonate salt at pH 7 and pH 7.5, respectively. The proposed chemiluminescence reaction mechanism of acridinium ester via a dioxetanone structure was evaluated via quantum chemical calculation on density functional theory. The proposed mechanism was composed of the nucleophilic addition reaction of hydroperoxide anion, dioxetanone ring formation, and nonadiabatic transition due to spin-orbit coupling around the transition state (TS) to the triplet state (T1) following the decomposition pathway. The TS which appeared in the thermal decomposition would be a rate-determining step for all three processes.

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 Zn(2+) 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 Mg(2+), Mn(2+) and Ca(2+) produced virtually no change in the quantum yields or the spectra of bioluminescence. In contrast, increasing amounts of ions such as Zn(2+) and Cd(2+) 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 Hg(2+) >Zn(2+), Cd(2+) >Ni(2+), Co(2+), Fe(2+) ≫Mg(2+), Mn(2+), Ca(2+). 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.

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.

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.