Spectroscopic studies of Delayed Luminescence emitted by an in vitro model for studying of the effects of amyloid-βeta (Aβ) have been performed. Aβ is a neurotoxic protein overexpressed in Alzheimer's Disease (AD), which is also related to mitochondrial dysfunction. The experiments have been carried out on primary Olfactory Ensheathing Cells (OECs) cultures. Cells have been exposed to Aβ(1-42) native full-length peptide or to Aβ(25-35), a toxic fragment of Aβ, or Aβ(35-25), a non toxic Aβ fragment, both in absence and in presence of Astaxanthin, a well-known antioxidant agent. To monitor cell viability, MTT test was used. Reactive oxygen species and reduced glutathione levels were utilized to test the oxidative intracellular status. We also assessed the expression of some glial markers (Glial Acidic Fibrillary Protein, Vimentin), of Nestin, stem cell marker, and the activation of the apoptotic pathway assessing caspase-3 cleavage. We found that, in OECs, Glial Acidic Fibrillary Protein, Vimentin expression and caspase-3 exhibited a significant enhancement in Aβ(1–42) and Aβ(25–35) exposed cells. The pre-treatment with Astaxanthin restored the levels of Vimentin and caspase-3 to control values, increasing also Nestin expression levels and reestablished the intracellular oxidative status modified by the exposure to Aβ(1–42) or Aβ(25–35) of OECs. DL intensity and kinetics changes as a function of the treatments were also measured. In particular, an increase in DL emission, with respect the untreated cells (controls), was observed in cells exposed to Aβ(25-35) fragment. This emission appeared quenched in presence of Astaxanthin.
In the framework of the research project ETHICS “Pre-clinical experimental and theoretical studies to improve treatment and protection by charged particles” funded by the National Nuclear Physics Institute, Italy, we studied the phenomenon called delayed luminescence emitted by non-tumorigenic breast epithelial MCF10A cell line after proton irradiation at different doses (0.5, 2, 6, 9 Gy). The aim is to found possible correlations between delayed luminescence and in vitro damaging induced by ion irradiation. The first results of this research show that the delayed luminescence kinetics is proton dose dependent. An interesting correlation between delayed luminescence and clonogenic potential was observed.
The first results concerning the possibility to use Delayed Luminescence spectroscopy to evaluate the in vitro induction of cytotoxic effects on human glioblastoma cells of nanostructured lipid carrier and drug-loaded nanostructured lipid carrier are showed in this contribution. We tested the effects of nanostructured lipid carrier, ferulic acid and ferulic acidloaded nanostructured lipid carrier on U-87MG cell line. The study seems to confirm the ability of Delayed Luminescence to be sensible indicator of alterations induced on functionality of the mitochondrial respiratory chain complex I in U-87MG cancer cells when treated with nanostructured lipid carriers.
Correlation between apoptosis and UVA-induced ultraweak photon emission delayed luminescence (DL) from tumor thyroid cell lines was investigated. In particular, the effects of berberine, an alkaloid that has been reported to have anticancer activities, on two cancer cell lines were studied. The FTC-133 and 8305C cell lines, as representative of follicular and anaplastic thyroid human cancer, respectively, were chosen. The results show that berberine is able to arrest cell cycle and activate apoptotic pathway as shown in both cell lines by deoxyribonucleic acid fragmentation, caspase-3 cleavage, p53 and p27 protein overexpression. In parallel, changes in DL spectral components after berberine treatment support the hypothesis that DL from human cells originates mainly from mitochondria, since berberine acts especially at the mitochondrial level. The decrease of DL blue component for both cell lines could be related to the decrease of intra-mitochondrial nicotinamide adenine dinucleotide and may be a hallmark of induced apoptosis. In contrast, the response in the red spectral range is different for the two cell lines and may be ascribed to a different iron homeostasis.
Irina Baran, Diana Ionescue, Simona Privitera, Agata Scordino, Maria Mocanu, Francesco Musumeci, Rosaria Grasso, Marisa Gulino, Adrian Iftime, Ioana Tofolean, Alexandru Garaiman, Alexandru Goicea, Ruxandra Irimia, Alexandru Dimancea, Constanta Ganea
The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission [delayed luminescence (DL)] of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMN ox ) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMN ox . Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predominantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 μs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ.
Today, single photon imaging represents one of the most challenging goals in the field of photonics. Many areas are
involved: nuclear and particle physics, astronomy, and, in the biophysics field, the newest technique to investigate the
state of several biological systems by detecting the ultra-weak luminescence emitted from the excited sample under
study. Aim of the work is the realization of a single photon imaging device able to identify the position and the arrival
time of the impinging photons from ultra low intensity sources. The main features of a 2-D array of Single Photon
Avalanche Diodes, manufactured by ST-Microelectronics, are shown.
KEYWORDS: Skin, In vivo imaging, Luminescence, Data modeling, Single photon, Tissues, Fluorescence spectroscopy, Biomedical optics, Spectroscopy, Solids
The UVA induced Delayed Luminescence (DL), has been measured in vivo in the forearm skin of some healthy
volunteers of different sex and age during several periods of the year. An innovative instrument able to detect, in single
photon counting mode, the spectrum and the time trend of the DL emission has been used. The measured differences in
the time trends of the spectral components may be related to the sex and the age. The potential development of a new
analysis technique based on this phenomenon is discussed.
In this contribution we describe the realization of MUSES, a novel research equipment able to detect and identify
photons emitted, after laser irradiation, from biological samples (like micro-organisms and human cells) for fast
ultraweak luminescence analysis. MUSES has been entirely designed and realised at LNS-Southern National Laboratory
of the Italian INFN-Nuclear Physics National Institute. The excellent performances in terms of timing, wavelength and
angular identification make this multi-detector a unique device in biophotonics research field.
The new developments of SINPHOS project (SINgle PHOton Spectrometer) are reported. The realised device
is able to measure simultaneously with high accuracy time distribution and the wavelength spectrum of photons coming
several physical and biological systems. Such device is essentially composed by a grading spectrometer and an array of
SPADs (Single Photon Avalange Diodes).
Design and characterization of a new generation of single photon avalanche diodes (SPAD) array, manufactured by STMicroelectronics
in Catania, Italy, are presented. Device performances, investigated in several experimental conditions
and here reported, demonstrate their suitability in many applications. SPADs are thin p-n junctions operating above the
breakdown condition in Geiger mode at low voltage. In this regime a single charged carrier injected into the depleted
layer can trigger a self-sustaining avalanche, originating a detectable signal. Dark counting rate at room temperature is
down to 10 s-1 for devices with an active area of 10 μm in diameter, and 103 s-1 for those of 50 &mgr;m. SPAD quantum
efficiency, measured in the range 350÷1050 nm, can be comparable to that of a typical silicon based detector and reaches
the values of about 50% at 550 nm for bigger samples. Finally, the low production costs and the possibility of integrating
are other favorable features in sight of highly dense integrated 1-D or 2-D arrays.
New single photon avalanche detectors (SPAD), are presented. Device performances, as photo-detection efficiency, timing and dark counts, extracted in several experimental conditions and here reported, make them suitable in many applications. The integration possibility, in order to achieve a new concept of solid state photomultiplier, has been also successfully investigated within the 5x5 arrays manufacture.
Photobiological research in the last decades has shown the existence of Delayed Luminescence in biological tissue, which presents an excitation spectrum with a peak within the UVA region and can be detected with sophisticated photomultiplier systems. Based on these findings, a new and powerful tool able to measure the UV-A-laser-induced Delayed Luminescence emission of cultured cells was developed, with the intention to detect biophysical changes between carcinogenic and normal cells. Indeed noticeable differences have been found in the time resolved emission spectrum of delayed luminescence of cell cultures of human fibroblast and human melanoma. This new, powerful and non-invasive technique, in principle, could be applied in all fields of skin research, such as the investigation of skin abnormalities and to test the effect of products involved in regeneration, anti-aging and UV-light protection in order to prevent skin cancer.
SINPHOS is a monolithic micro-device, able to measure simultaneously time distribution and spectrum of photons coming from a weak source like Delayed Luminescence of biological systems. In order to achieve this challenging goal, we use: Deep Lithography with Ions (DLI) and microelectronic technologies for the fabrication of dedicated passive micro-optical elements and for the realization of Single Photon Avalanche Diode (SPAD) detectors, respectively
Photobiological research in the last 30 yr has shown the existence of ultraweak photon emission in biological tissue, which can be detected with sophisticated photomultiplier systems. Although the emission of this ultraweak radiation, often termed biophotons, is extremely low in mammalian cells, it can be efficiently increased by ultraviolet light. Most recently it was shown that UV-A (330 to 380 nm) releases such very weak cell radiation in differentiated human skin fibroblasts. Based on these findings, a new and powerful tool in the form of UV-A-laser-induced biophotonic emission of cultured cells was developed with the intention to detect biophysical changes between carcinogenic and normal cells. With suspension densities ranging from 1 to 8×106 cells/mL, it was evident that an increase of the UV-A-laser-light induced photon emission intensity could be observed in normal as well as melanoma cells. Using this new detection procedure of ultraweak light emission, photons in cell suspensions as low as 100 µL could be determined, which is a factor of 100 lower compared to previous procedures. Moreover, the detection procedure has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 µs instead of 150 ms, as reported in previous procedures. This improvement leads to measurements of light bursts up 107 photons/s instead of several hundred as found with classical designs. Overall, we find decreasing induction ratings between normal and melanoma cells as well as cancer-prone and melanoma cells. Therefore, it turns out that this highly sensitive and noninvasive device enables us to detect high levels of ultraweak photon emission following UV-A-laser-induced light stimulation within the cells, which enables future development of new biophysical strategies in cell research.
Delayed Luminescence is a well established technique based on the illumination of biological sample and on the subsequent count of the number of photons re-emitted by the sample after the light source has been switched off together with their spectral distribution. Investigations have been performed on yeast cells and algae so that correlations can be established between biological activities and physical parameters of the samples. Moreover nonlinear mechanisms of interaction between optical fields and cells can be figured out. Further investigations will be reported on yeast cell samples deposited on paper filters after irradiation by soft X-rays. The results will be discussed by cross correlating the experimental evidence from Delayed Luminescence with those obtained by metabolic activity recording. Luminescence and Delayed Luminescence are strictly correlated with early insurgence of morphological alterations of normal or pathological nature in cells and tissues; a novel technique for morphological analysis has been developed by means of Focused Ion Beam machines. A straightforward approach to morphology at the nanoscale both of membranes and cellular inner structures is then made possible. The final aim is an experimental set up for an early and reliable detection technique for neoplastic cells and tissues sorting.
Delayed luminescence (D.L.) is a measure that provides important information on biological systems fields, structures and activities, by counting impinging and emitted photons. Many recent experimental works have shown the existence of a close connection, sometimes analytically expressed between the biological state of the system and D.L. parameters. Our investigations aim to show that D.L. is a workable analytical technique covering a large number of disciplinary fields, from agriculture to pollution control and from medical diagnostics to food quality control. The authors have conducted systematic research about D.L. from unicellular alga Acetabularia acetabulum to Saccharomyces cerevisiae yeast cultures and about more complex systems such as Soya seed (Glycine max, L.) and its dependence on sample preparation, history, intracellular signaling, metabolism and pollutant presence. We will discuss the most relevant results together with theoretical considerations on the basic interaction at work between biological systems and electromagnetic fields.
Ultraweak biophoton emission has been recorded in synchronously developing populations of early Drosophila embryos. On being stimulated briefly with white light within the first 40 minutes of development, a new kind of luminescence is observed in the form of superdelayed, intense flashes. We assume that the initial light stimulation sets up some kind of coherent interaction inside the system and develop a phenomenological number-phase-angle model to give a general interpretation of the superdelayed radiation patterns.
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