Ready vascular permeability of a near-infrared fluorescent agent ASP5354 for intraoperative ureteral identification enables imaging of carcinoma tissues.

This study investigates the ability of a near-infrared fluorescence (NIRF) imaging agent, ASP5354, for in vivo fluorescence imaging of esophageal squamous cell carcinoma (ESCC) tissues. The ability of ASP5354 was evaluated using a single dose of ASP5354 or indocyanine green (ICG), which was intravenously administered to a KYSE850 human ESCC xenograft mouse model. Subsequently, in vivo NIRF images of the mouse were obtained using a clinically available camera system. ASP5354-specific NIRF signals were strongly detectable in KYSE850 carcinoma tissues immediately (30 s) following ASP5354 administration compared with normal tissues. Meanwhile, ICG could not distinguish between normal and carcinomatous tissues. To elucidate the associated imaging mechanisms, the vascular permeability of ASP5354 and ICG was investigated in rat back dermis treated with saline or histamine, which enhances vascular permeability, using in vivo NIRF imaging. ASP5354 exhibited higher vascular permeability in histamine-treated skin than in normal skin. KYSE850 carcinoma tissues can be distinguished from normal tissues based on the measurement of ASP5354-specific NIRF signals, and the mechanism that enables imaging relies on the specific and rapid leakage of ASP5354 from the capillaries into the stroma of carcinoma tissues.

Evaluation of the Utilization of Near-Infrared Fluorescent Contrast Agent ASP5354 for In Vivo Ureteral Identification in Renal Diseases Using Rat Models of Gentamicin-Induced Acute Kidney Injury.

ASP5354 was recently developed as a near-infrared fluorescence (NIRF) contrast agent for intraoperative ureteral identification, and its use has been evaluated in healthy animals. However, the utilization of ASP5354 for ureteral identification has not been evaluated in animals with renal injury. In this study, we assessed the application of ASP5354 for ureteral imaging using rat models of gentamicin-induced mild, moderate, and severe acute kidney injury (AKI), using a clinically available NIRF detection system. NIRF was detected in the abdominal cavity and ureters after laparotomy, and the efficiency of ASP5354 was evaluated based on the NIRF signal intensity over 60 min. After the intravenous injection of ASP5354 into rats with mild or moderate AKI, the ureters were clearly imaged at a high ratio of NIRF intensity in the ureter to that in the tissues around the ureter. Six days after intravenous injection, the use of ASP5354 in rats with moderate AKI did not affect the biochemical kidney functions or histopathological conditions of the kidney tissues, as compared to those with no injection of ASP5354. In rats with severe AKI, ureteral imaging was not effective due to the relatively strong NIRF expression in the tissues around the ureters. These data indicate that ASP5354 holds potential as a contrast agent for intraoperative ureteral identification in patients with limited renal injury.

In vivo near-infrared fluorescence imaging of gastric cancer in an MKN-45 gastric cancer xenograft mouse model using intraoperative ureteral identification agent ASP5354.

Accurate intraoperative identification of gastric cancer lesions and determination of the extent of resection are important for curability and function preservation. This study aimed to investigate the potential of the near-infrared fluorescence (NIRF) imaging agent ASP5354 for in vivo fluorescence imaging of gastric cancer. The capability of ASP5354 was evaluated using an MKN-45 human gastric cancer xenograft mouse model. A single dose of ASP5354 was intravenously administered to the mice at a concentration of 120 nmol (0.37 mg)/kg body weight. In vivo NIRF images of the mouse backs were obtained using an NIRF camera system. Moreover, the cancer tissues were dissected, and the NIRF intensity in the tissue sections was measured using the NIRF camera system. ASP5354 uptake in MKN-45 cells was assessed in vitro using the NIRF microscope. The NIRF signal of ASP5354 was selectively detected in gastric cancer tissues immediately after the intravenous administration of ASP5354. The cancer tissues emitted stronger NIRF signals than adjacent normal tissues. The difference in the NIRF intensity between the normal and cancer tissues was clearly observed at the boundary between them in the macrolevel NIRF images. Cancer tissues can be distinguished from normal tissues based on the measurement of the NIRF of ASP5354, using an NIRF camera system. ASP5354 is a promising agent for NIRF imaging of gastric cancer tissues.

In Vivo Optical Imaging of Bladder Cancer Tissues in an MB49 Bladder Cancer Orthotopic Mouse Model Using the Intravesical or Intravenous Administration of Near-Infrared Fluorescence Probe.

Bladder cancer was the twelfth most common cancer worldwide in 2020. Although bladder cancer has been diagnosed using macroscopic techniques, such as white-light cystoscopy and fluorescence blue-light cystoscopy, there is a need to explore more effective noninvasive optical imaging techniques for accurate bladder cancer diagnosis. This study demonstrates the high effectiveness of the near-infrared fluorescence (NIRF) probe ASP5354, which has been developed for ureteral identification during in vivo diagnosis of bladder cancer in an MB49 bladder cancer orthotopic mouse model. After the intravesical injection of 2.4 µM ASP5354 followed by bladder rinsing with saline at 5 min post injection or intravenous administration of ASP5354 at 240 nmol/kg mouse body weight, followed by a waiting period of 5-24 h in mice, ASP5354 was absorbed specifically by cancerous tissue and not by normal tissues in the bladder. NIRF of ASP5354 in cancer tissues was detected using the NIRF imaging camera system. The NIRF clearly showed a boundary between cancerous and normal tissues. Therefore, ASP5354 provides noninvasive and specific optical in vivo imaging of MB49 bladder cancer using intravesical or intravenous injection of ASP5354. ASP5354 may allow for new diagnostic applications for bladder cancer in humans.

Near-Infrared Fluorescence Imaging of Renal Cell Carcinoma with ASP5354 in a Mouse Model for Intraoperative Guidance.

Renal cell carcinoma is a prevalent disease associated with high morbidity and mortality rates. Partial nephrectomy is a first-line surgical option because it allows the preservation of renal function. Clear differentiation between normal and cancerous tissues is critical for increasing the negative margin rates. This study investigated the capability of the near-infrared (NIR) fluorescent imaging agent ASP5354 for in vivo fluorescence imaging of renal cell carcinoma. ASP5354 at a single dose of 12 nmol (0.037 mg)/kg body weight was intravenously administered to healthy and orthotopic renal cell carcinoma mice under anesthesia. NIR images of the abdominal cavity were obtained using a near-infrared fluorescence (NIRF) camera system. In addition, the cancerous kidneys were harvested, and the NIRF in their sections was measured using an NIRF microscope. Normal renal tissue emitted strong NIRF but the cancer tissue did not. The difference in NIRF intensity between the normal and cancer tissues clearly presented the boundary between the normal and cancer tissues in macro and micro NIRF imaging. ASP5354 can distinguish cancer tissue from normal tissue using NIRF. Thus, ASP5354 is a promising agent for renal cell carcinoma tissue imaging in partial nephrectomy for renal cell carcinoma patients.

Near-infrared chemiluminescence imaging of superoxide anion production in kidneys with iron3+-nitrilotriacetate-induced acute renal oxidative stress in rats.

Iron-catalyzed oxidative stress generates reactive oxygen species in the kidney and induces oxidative damage including lipid, protein, and DNA modifications which induces renal injury and may lead to cancer. An analysis of oxidative stress dynamics by reactive oxygen species has not been performed non-invasively in real time in intact kidneys and is a significant challenge in biology and medicine. Here, I report that MCLA-800 is a near-infrared chemiluminescent probe that visualizes the dynamics of superoxide anion (O2•-) production and the upstream generation of reactive oxygen species in living rat kidneys suffering acute renal oxidative stress induced by intraperitoneal administration of iron3+-nitrilotriacetate (Fe3+-NTA) as a representative Fe3+ chelate. MCLA-800 was intravenously injected at 250 nmol/kg body weight and immediately transported to the kidneys with the emitting light dependent on O2•- production. The magnitude of O2•- production correlated with the Fe3+-NTA dose. O2•- was continuously produced in the blood stream following Fe3+-NTA injection at 0.15 mmol/kg body weight, while peak production in the renal cortex occurred at 24 h, then decreased to the background level at 72 h. This study clearly revealed the dynamics of Fe3+-NTA-mediated O2•- production in the living kidney by chemiluminescent imaging of O2•- production using MCLA-800.

Non-invasive and accurate readout of superoxide anion in biological systems by near-infrared light.

Infectious and inflammatory diseases involve superoxide anion (O2•-) production. Real-time and non-invasive evaluation of O2•- in intact biological systems has been a significant challenge in biology and medicine. Here, I report that an advanced near-infrared chemiluminescent probe, MCLA-800, enables reliable non-invasive optical readout of O2•-ex vivo and in vivo. MCLA-800 allowed highly selective and sensitive monitoring of O2•- in undiluted human whole blood ex vivo. For the first time, the use of MCLA-800 revealed two reproducible types of O2•- production in response to stimulation by unopsonized zymosan particles of Saccharomyces cerevisiae, that is, slow response (S-type) and fast response (F-type), specific to each individual. O2•- production was synchronized with myeloperoxidase (MPO) activation in the former type but not in the latter. Moreover, as new findings, MCLA-800 chemiluminescence demonstrated that the chemiluminescence intensity-time properties of formyl-methionyl-leucyl-phenylalanine (fMLP)- or phorbol 12-myristate 13-acetate (PMA)-induced O2•- production and MPO activity were independent of S- and F-type zymosan-induced MCLA-800 chemiluminescence whole blood and that PMA-induced MPO activation synchronized with PMA-induced O2•- production in S- and F-type zymosan-induced MCLA-800 chemiluminescence whole blood, but fMLP-induced MPO activation did not synchronize with fMLP-induced O2•- production in both of S- and F-type blood. Furthermore, MCLA-800 spatiotemporally allowed non-invasive and clear in vivo imaging of O2•- in animal models of acute dermatitis and focal arthritis. Therefore, MCLA-800 could be possibly applied in various advanced diagnostic techniques.

A Near-Infrared Fluorescent Probe Coated with ß-Cyclodextrin Molecules for Real-Time Imaging-Guided Intraoperative Ureteral Identification and Diagnosis.

Although iatrogenic ureteral injury and its lack of recognition due to ureteral invisibility are serious incidents in open and laparoscopic abdominal surgeries, there are currently no safe and effective methods for intraoperative ureteral identification (IUI) and diagnosis (IUD). In this study, I designed and chemically synthesized a near-infrared fluorescence (NIRF) imaging probe (CD-NIR-1) and evaluated its clearance and ability for IUI and IUD in animal models. CD-NIR-1 demonstrated high specificity and ultrarapid clearance by rat kidneys to the urinary bladder following intravenous administration of a single dose (25 nmol/kg of body weight), with 96% of the dose ultimately excreted at the first urination with no chemical modification. Furthermore, urine containing CD-NIR-1 in ureters showed strong NIRF, thereby enabling IUI and IUD via NIRF imaging. These results demonstrated the efficacy of CD-NIR-1 for clinical use.

Prediction and suppression of internal blue discoloration in roots of daikon, the Japanese radish (Raphanus sativus L.).

The internal blue discoloration of edible daikon roots often occurs on day 3 after harvest during storage at 20°C and is a serious problem. This study reports a rapid and simple method for predicting discoloration at harvest and proposes two methods for suppressing the discoloration of roots that are at discoloration risk. The soaking of freshly harvested roots in aqueous hydrogen peroxide resulted in immediate blue discoloration. The correlation between discoloration after storage at 20°C and discoloration after soaking in hydrogen peroxide was positive. Discoloration using hydrogen peroxide at harvest is a useful way of predicting discoloration risk. The storage of roots at 10°C in air or at 20°C in an atmosphere containing 1% (v/v) molecular oxygen resulted in no discoloration for at least 8 days. These storage conditions can guarantee no discoloration for the distribution after harvest.

Trans-3-hydroxyhispidin is not an actual bioluminescence substrate in pileus gills of the luminous fungus Mycena chlorophos.

Mycena chlorophos is a species of molecular oxygen-dependent bioluminescent fungus, and its pileus gills emit bright green light. The chemical mechanisms underlying this bioluminescence phenomenon are not yet understood. An enzyme (luciferase) producing light from trans-3-hydroxyhispidin is present in M. chlorophos pileus gills. However, it is unclear whether trans-3-hydroxyhispidin is an actual bioluminescence substrate (luciferin) in the natural bioluminescence of M. chlorophos. In the present study, this question is resolved. It was clearly demonstrated that the trans-3-hydroxyhispidin analog trans-3-hydroxybisnoryangonin significantly inhibited the artificial luminescence induced by the addition of trans-3-hydroxyhispidin to living pileus gills but did not inhibit natural bioluminescence in living pileus gills. This inhibition was due to the reaction of trans-3-hydroxybisnoryangonin with luciferase for trans-3-hydroxyhispidin. Even though trans-4-aminocinnamic acid is known to inhibit natural bioluminescence in living pileus gills, in the present study, trans-4-aminocinnamic acid did not influence the artificial luminescence via trans-3-hydroxyhispidin in the presence of luciferase for trans-3-hydroxyhispidin. These inconsistencies between the natural bioluminescence and the artificial luminescence of trans-3-hydroxyhispidin indicate that trans-3-hydroxyhispidin is not an actual luciferin in natural bioluminescence. Trans-3,4-dihydroxycinnamic acid is generally known to be an intermediate in trans-3-hydroxyhispidin biosynthesis. The artificial luminescence induced by the addition of trans-3,4-dihydroxycinnamic acid to living pileus gills was not inhibited by trans-3-hydroxybisnoryangonin. Therefore, trans-3,4-dihydroxycinnamic acid does not contribute to the luminescence involving trans-3-hydroxyhispidin in living pileus gills.

Bioluminescence and chemiluminescence abilities of trans-3-hydroxyhispidin on the luminous fungus Mycena chlorophos.

The fungus Mycena chlorophos emits green light from its pileus gills but not from its stipes. The chemical mechanisms underlying its bioluminescence are unclear. Trans-3-hydroxyhispidin has been known to be a luminescence substrate for the bioluminescent mycelia of Neonothopanus nambi and N. gardneri. In the present study, the bioluminescence and chemiluminescence abilities of trans-3-hydroxyhispidin on pileus gills and originally non-bioluminescent stipes of M. chlorophos were demonstrated. Trans-3-hydroxyhispidin induced bioluminescence of living gills and stipes. The bioluminescence spectra of living gills and stipes measured after the addition of trans-3-hydroxyhispidin were consistent with the original bioluminescence spectrum of gills. Frozen-thawed (dead) gills and stipes maintained trans-3-hydroxyhispidin luminescence activity, and the luminescence-active enzyme (luciferase) was partially purified in the water-insoluble state from both tissues using gel filtration followed by ultracentrifugation. The optimum temperature of the chemiluminescence reactions of trans-3-hydroxyhispidin in the presence of partially purified gill or stipe luciferase was 25°C. The chemiluminescence quantum yields of trans-3-hydroxyhispidin for gill luciferase and stipe luciferase in 20 mM phosphate buffer at 25°C were 0.017 and 0.00096, respectively and the chemiluminescence spectra were almost consistent with the bioluminescence spectrum of living gills. These results indicate that trans-3-hydroxyhispidin can be a candidate as a substrate for M. chlorophos bioluminescence.

Inhibition of bioluminescence in the living gills of the luminous fungus Mycena chlorophos by trans-4-aminocinnamic acid.

The living gills of the fungus Mycena chlorophos spontaneously emit green light. It was previously reported that trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnnamic acid are essential for the bright light production in the living gills. However, the chemical mechanisms underlying their bioluminescence are unknown. In the present study, trans-4-aminocinnamic acid was found to inhibit light production in the living gills. The concentrations of trans-4-aminocinnamic acid that inhibited the bioluminescence intensity by 50% of initial values for immature and mature gills were 0.07 µM and 4 µM, respectively. Approximately 20% of the bioluminescence intensity of the immature and mature gills was not inhibited by a further increase in the concentration of trans-4-aminocinnamic acid. Moreover, the bioluminescence that was activated by trans-4-hydroxycinnamic acid or trans-3,4-dihydroxycinnamic acid (0.01 mM) was completely inhibited by trans-4-aminocinnamic acid. Therefore, trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid functioned for the bioluminescence that was inhibited by trans-4-aminocinnamic acid. trans-4-Aminocinnamic acid strongly bound to the bioluminescence system(s) and withstood rinsing of the gills with 10 mM phosphate buffer (pH = 7), and high concentrations of trans-4-hydroxycinnamic acid (1 mM) and trans-3,4-dihydroxycinnamic acid (0.1 mM) functioned to displace trans-4-aminocinnamic acid from the bioluminescence system(s) and reactivate bioluminescence. Benzenamine, trans-cinnamic acid, trans-2-aminocinnamic acid, and trans-3-aminocinnamic acid did not inhibit bioluminescence. Therefore, the structure-specific inhibition by trans-4-aminocinnamic acid suggested that the 4-hydroxy group in trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid molecules plays a functional role in the bioluminescence reaction.

A combination of NADHP and hispidin is not essential for bioluminescence in luminous fungal living gills of Mycena chlorophos.

The chemical mechanisms underlying visible bioluminescence in the fungus Mycena chlorophos are not clear. A combination of dihydronicotinamide adenine dinucleotide phosphate (NADPH) and hispidin, which has been reported to increase the intensity of in vitro luminescence in crude cold-water extracts prepared from the bioluminescent fruiting bodies of M. chlorophos, exhibited potential bioluminescence activation in the early bioluminescence stages, in which the bioluminescence was ultra-weak, for living gills and luminescence activation for non-bioluminescent gills, which was collapsed by freezing and subsequent thawing, at all bioluminescence stages. These abilities were not evident in considerably bioluminescent gills. These abilities were blocked by trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid, which were identified as in vivo bioluminescence-activating components. Original bioluminescence and bioluminescence produced from the addition of trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnamic acid in living gills were almost completely inhibited by 10 mM NaN3 , whereas the luminescence produced form the combination of NADPH and hispidin in thawed non-bioluminescent and living gills at the early weak bioluminescence stages was not inhibited by 10 mM NaN3 . Thus, the combination of NADPH and hispidin plays different roles in luminescence systems compared with essential bioluminescence systems, and the combination of NADPH and hispidin was not essential for visible bioluminescence in living gills.

Second bioluminescence-activating component in the luminous fungus Mycena chlorophos.

Mycena chlorophos is an oxygen-dependent bioluminescent fungus. The mechanisms underlying its light emission are unknown. A component that increased the bioluminescence intensity of the immature living gills of M. chlorophos was isolated from mature M. chlorophos gills and chemically characterized. The bioluminescence-activating component was found to be trans-3,4-dihydroxycinnamic acid and its bioluminescence activation was highly structure-specific. 13 C- and 18 O-labelling studies using the immature living gills showed that trans-3,4-dihydroxycinnamic acid was synthesized from trans-4-hydroxycinnamic acid in the gills by hydroxylation with molecular oxygen as well as by the general metabolism, and trans-3,4-dihydroxycinnamic acid did not produce hispidin (detection-limit concentration: 10 pmol/1 g wet gill). Addition of 0.01 mM hispidin to the immature living gills generated no bioluminescence activation. These results suggested that the prompt bioluminescence activation resulting from addition of trans-3,4-dihydroxycinnamic acid could not be attributed to the generation of hispidin. Copyright © 2016 John Wiley & Sons, Ltd.

Near-infrared indocyanine dye permits real-time characterization of both venous and lymphatic circulation.

We investigated the optical properties of a near-infrared (NIR) fluorochrome, di-ß-cyclodextrin-binding indocyanine derivative (TK-1), and its pharmacokinetic differences with indocyanine green (ICG). TK-1 was designed to have hydrophilic cyclodextrin molecules and, thus, for higher water solubility and smaller particle sizes than the plasma protein-bound ICG. We compared optical properties such as the absorption and fluorescence spectra, quantum yield, and photostability between both dyes in vitro. In addition, we subcutaneously injected a 1 mM solution of TK-1 or ICG into the hind footpad of rats and observed real-time NIR fluorescence intensities in their femoral veins and accompanying lymphatics at the exposed groin site to analyze the dye pharmacokinetics. These optical experiments demonstrated that TK-1 has high water solubility, a low self-aggregation tendency, and high optical and chemical stabilities. Our in vivo imaging showed that TK-1 was transported via peripheral venous flow and lymphatic flow, whereas ICG was drained only through lymphatics. The results of this study showed that lymphatic and venous transport can be differentially regulated and is most likely influenced primarily by particle size, and that TK-1 can enable real-time NIR fluorescence imaging of whole fluids and solute movement via both microvessels and lymphatics, which conventional ICG cannot achieve.

Mechanism Underlying the Onset of Internal Blue Discoloration in Japanese Radish (Raphanus sativus) Roots.

The internal blue discoloration observed in Japanese radish (Raphanus sativus L.) roots is a physiological phenomenon caused by storage following harvest at approximately 20 °C and poses a serious problem for farmers. Here, we describe the mechanism underlying the onset of internal blue discoloration of three cultivars: Hukuhomare, SC8-260, and Yuto. Each cultivar was maintained under the same conditions. Additionally, Hukuhomare radish roots were maintained at three different cultivation conditions in a related experiment. The blue discoloration in radish roots was caused by the oxidation of 4-hydroxyglucobrassicin as a result of an increase in oxidative stress involving peroxidase. Thus, the extent of blue discoloration was influenced by the chemical balance involving 4-hydroxyglucobrassicin content, antioxidant capacity, and oxidation activity.

Structure of a Precursor to the Blue Components Produced in the Blue Discoloration in Japanese Radish (Raphanus sativus) Roots.

The internal blue discoloration in Japanese radish (Raphanus sativus L.) roots has been reported to be a physiological phenomenon after harvest and poses a significant problem for farmers. To avoid this discoloration, the fundamental development of new radish cultivars that do not undergo discoloration and/or improved cultivation methods is required. Elucidating the chemical mechanism leading to this discoloration could help overcome these difficulties. To determine the mechanism underlying this discoloration, this study was designed to probe the structure of a precursor to the blue components generated during the discoloration process. Soaking fresh roots in aqueous H2O2 resulted in rapid blue discoloration, similar to the natural discoloration. Using a H2O2-based blue discoloration assay, the precursor was extracted and isolated from the fresh roots and identified as the glucosinolate, 4-hydroxyglucobrassicin, via spectroscopy and chemical synthesis.

Identification of possible light emitters in the gills of a bioluminescent fungus Mycena chlorophos.

The pileus of Mycena chlorophos actively, spontaneously, and continuously emits green light. Molecular mechanisms underlying this bioluminescence remain unclear. We investigated light emitters in the pileus of M. chlorophos to determine the underlying mechanisms. High-performance liquid chromatography-fluorescence-photodiode array-mass detection analyses showed that actively luminescent gills in the pileus exclusively and abundantly possessed riboflavin, riboflavin 5'-monophosphate, and flavin adenine dinucleotide as green-fluorescent components. These components were localized in the bioluminescent region of the gills at the microscopic level. Fluorescence spectra of these green-fluorescent components and the gills were identical with the spectrum of gill bioluminescence (maximum emission wavelength, 525 nm). Thus, our results indicated that the possible light emitters in the pileus of M. chlorophos were riboflavin, riboflavin 5'-monophosphate, and/or flavin adenine dinucleotide. Copyright © 2016 John Wiley & Sons, Ltd.

Localization of the bioluminescence system in the pileus of Mycena chlorophos.

Mycena chlorophos, which is primarily distributed in Southeast Asia, is a luminous fungus that emits a bright green light from its pileus for about 2 days at approximately 20°C and high relative humidity. The distribution of bioluminescent tissues in the whole pileus and its sections was heterogeneous. The light intensity in the cap and the upper region of the gill was greater than that in the lower region of the gill. At the microscopic level, the light was predominantly emitted from the membranes of hymenium and basidia cells on the gill. The emission was both cell and region specific. The luminescence system was localized in the cell membrane, and a part of the system was on the cell membrane surface.

Bioluminescence of the arm light organs of the luminous squid Watasenia scintillans.

The squid Watasenia scintillans emits blue light from numerous photophores. According to Tsuji [F.I. Tsuji, Bioluminescence reaction catalyzed by membrane-bound luciferase in the "firefly squid", Watasenia scintillans, Biochim. Biophys. Acta 1564 (2002) 189-197.], the luminescence from arm light organs is caused by an ATP-dependent reaction involving Mg2+, coelenterazine disulfate (luciferin), and an unstable membrane-bound luciferase. We stabilized and partially purified the luciferase in the presence of high concentrations of sucrose, and obtained it as particulates (average size 0.6-2 microm). The ATP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the particulate luciferase was investigated in detail. Optimum temperature of the luminescence reaction is about 5 degrees C. Coelenterazine disulfate is a strictly specific substrate in this luminescence system; any modification of its structure resulted in a very heavy loss in its light emission capability. The light emitter is the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate is calculated at 0.36. ATP could be replaced by ATP-gamma-S, but not by any other analogues tested. The amount of AMP produced in the luminescence reaction was much smaller than that of coelenteramide disulfate, suggesting that the reaction mechanism of the Watasenia bioluminescence does not involve the formation of adenyl luciferin as an intermediate.