WordNet

relating to or exhibiting actinism

PrepTutorEJDIC

化学線の;化学作用のある

English Journal

Single-molecule photophysics of dark quenchers as non-fluorescent FRET acceptors.

Holzmeister P1, Wünsch B, Gietl A, Tinnefeld P.Author information 1TU Braunschweig, Institut für Physikalische und Theoretische Chemie, Hans-Sommer-Str. 10, 38106 Braunschweig, Germany. p.tinnefeld@tu-braunschweig.de.AbstractDark quencher chromophores are interesting alternatives to common single-molecule FRET acceptors. Due to their short excited state lifetime, they should be less prone to complex photophysics and bleaching. We find, however, that for common enzymatic oxygen scavenging systems and photoprotection strategies - the gold standard of single-molecule measurements - the quenchers BBQ650 and BHQ-2 induce frequent blinking of the donor molecule. They switch in a photoinduced process to what we identify as a radical anion state and back. We further make use of the broad absorption spectrum for selective bleaching of the quenchers in order to photoactivate the fluorescence of initially completely quenched dye molecules. This represents a general strategy to turn fluorescent dyes into photoactivatable probes.
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.Photochem Photobiol Sci.2013 Oct 7. [Epub ahead of print]
Dark quencher chromophores are interesting alternatives to common single-molecule FRET acceptors. Due to their short excited state lifetime, they should be less prone to complex photophysics and bleaching. We find, however, that for common enzymatic oxygen scavenging systems and photoprotection stra
PMID 24100609

The Proximal Priority Theory: An Updated Technique in Low Level Laser Therapy with an 830 nm GaAlAs Laser.

Ohshiro T.Author information Japan Medical Laser Laboratory, Shinanomachi, Shinjuku, Tokyo, Japan.AbstractBackground and Aims: The 830 nm GaAlAs diode laser has played an extremely active role in low level laser therapy (LLLT) since the early 1980's. Recently, the author modified his original proximal priority laser technique (PPLT), and the current article set out to explain the improved approach and show scientific evidence for its efficacy. Laser Therapy System: The laser therapy system used was based on the GaAlAs diode (OhLase-3D1, JMLL, Japan), delivering 60 mW in continuous wave at a wavelength of 830 nm in the near infrared with a power density at the tip of the probe head of approximately 1.2 W/cm(2). Proximal Priority Laser Technique: Under the author's PPLT concept, the brain is the control center for the body so every other part of the body is distal to the head. The main blood supply to the head is through the carotid arteries, and the deep penetration of the 830 nm beam applied to the side of the neck can involve and photoactivate the external and internal carotids, increasing the blood supply to the brain and creating a systemic parasympathetic system-mediated whole-body effect. The author has added gentle neck-stretching, trunk-stretching and his distal tissue softening approaches concomitant with the irradiation which enhance treatment efficacy. Results: Real-time fine-plate thermography has revealed whole-body warming as a result of the PPLT, with applications including chronic pain attenuation, female infertility and functional training of paraplegic cerebral palsy patients. The warming effect had a latency from hours to days, increasing in intensity and latency with subsequent PPLT sessions. Both Doppler flowmetry and SPECT have shown increased cerebral and systemic blood flow following PPLT. Conclusions: PPLT is easy to deliver and offers tangible results in a large range of conditions, enhancing the efficacy of diode laser LLLT.
Laser therapy.Laser Ther.2012 Dec 26;21(4):275-85. doi: 10.5978/islsm.12-OR-16.
Background and Aims: The 830 nm GaAlAs diode laser has played an extremely active role in low level laser therapy (LLLT) since the early 1980's. Recently, the author modified his original proximal priority laser technique (PPLT), and the current article set out to explain the improved approach and s
PMID 24511197

A faster, high resolution, mtPA-GFP-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy.

Lovy A1, Molina AJ, Cerqueira FM, Trudeau K, Shirihai OS.Author information 1Department of Neuroscience, Center for Neuroscience Research, Tufts School of Medicine.AbstractMitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment. Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary. Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment. A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP). Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE. The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity. In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15 nM TMRE in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.
Journal of visualized experiments : JoVE.J Vis Exp.2012 Jul 20;(65):e3991. doi: 10.3791/3991.
Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverte
PMID 22847388

Japanese Journal

T0201-1-1 せん断応力下における血管内皮細胞細胞膜タンパク質の運動の可視化([T0201-1]細胞の構造と流れにおけるマイクロ・ナノスケール解析(1))

川崎那緒人,島田知弥,島田勇祐,福井武和,工藤奨
年次大会講演論文集 : JSME annual meeting 2010(8), 129-130, 2010-09-04
Many biological procedures in endothelial cell are arranged by shear stress stimulation derived from blood flow. In the past study, the effect of shear stress stimulation on the lipid membrane fluidit …
NAID 110008701008