Looking Through The Microscope : What can we learn from Fluorescence Lifetime Measurements about Reactive Oxygen Species variations in Living Cells? Thank.

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1 Looking Through The Microscope : What can we learn from Fluorescence Lifetime Measurements about Reactive Oxygen Species variations in Living Cells? Thank you for giving me the opportunity to present my work today. [(This work was carried on in the university of Perpignan in south of France near the border with spain.) As (professor xxx ) it was just said, it concerns reactive oxygen species measurement in living cells and I will present you the results we obtained with our new methods. But first, I want to introduce you the reactive oxygen species. In living cells, mainly in the mitochondria, part of the oxygen used by the cell are transformed in reactive species such as anion superoxide then hydrogen peroxide, hydroxyl radical and subsequent radical species. Some of them are radical compounds other not. The reactive oxygen species are necessary for the good functioning of the cells, but when increased, they are harmful and found in high concentration in numerous disease. Anne-Cécile Ribou, Tareck Rharass, Jean Vigo, Jean-Marie Salmon Institut de Modélisation et d’Analyse en Géo-Environnement et Santé, University of Perpignan, F Perpignan, FRANCE.

2 Addition of sodium azide, NaN3 (50 µM) in cell culture
Reactive Oxygen Species Increase in living cells treated with Catalase Inhibitor Addition of sodium azide, NaN3 (50 µM) in cell culture 30 10 First, I will start with one of the experiments we performed. The catalase is an enzyme found in the cells that regulate and decrease the concentration of hydrogen peroxide. It transform the hydrogen peroxide in water, one can say, that it desactivate H2O2. The inhibitor will induce an accumulation of hydrogen peroxide within the cell and induce an augmentation of successive/(following radical species) reactive oxygen species. We had followed/monitor the production of ROS after treatment with the inhibitor. We can see an increase of the ROS production from the first hour of treatment. However at that time part of the cell still behave as the control and are not yet affected. After one day the ROS are increase by twofold. And at longer time we even found cells with a three-time increase of ROS. After this try to convince you that our new method allow to follow reactive oxygen species production in a long time observation, I will come to usual considerations about the probes.

3 Fluorescent probes for Reactive Oxygen Species detection
+ ROS - Chemiluminescent Probes : Luminol 3-aminophthalate * + ROS - “Inactivated” fluorescent probes : Fluorescent Molecule H2Molecule The actual fluorescent probes for ROS detection are all using the same mechanisms (procedure). It needs a chemical reaction between the probe and the reactive species and the compounds that result from the reaction is fluorescent, either directly in the case of chemi-luminescent probe or after excitation in the case of reduced molecules. Derivatives of dichlorofluorescein are the most employed probes in accordance to publication number. The system is sensitive to (depend on) the probe concentration which can be a problem considering the probe loading within living cells. This is a completely different phenomenon that occurs between oxygen and fluorescent probes. The oxygen molecule is well-known to deactivate the fluorescence of organic and also transition metal complexes. The phenomenon, so called dynamic quenching, increase the non radiative desactivation or the energy transfer from the excited states of the probe, and so change the time that the probe stay on this exited state. The probe is not chemically transformed on its ground-state but its fluorescence intensity and lifetime are changed because of additional deactivation processes. The interesting point is that the fluorescent lifetime of a probe do not depends on the probe concentration. Ten years ago I start to work with those oxygen probes, and particularly pyrene butyric acid, in living cells and later I try to explain the strange behaviors I observed in the living cells. (Dichlorofluorescein, dihydrorhodamine123 ou amplex Red). That mean the desactivation of the fluorescence of the probe will be accompanied of production of oxygen singulet mainly if the molecule excited phenomenom go through the triplet state and depending of probability consideration. This is a well-know phenomenom in case of porphirine phtosensibilsation in phototherapy. Fluorescent probes for Oxygen detection S0 S1 S2 T1 1A 1O2 3O2 1M Aromatic compound - Transition Metal complex 1- Pyrene Butyric Acid Tris(2,2’-bipyridyl)ruthenium(II)

4 Fluorescent Probes for Oxygen and Reactive Oxygen Species detection
Quantification of the quencher via the Stern-Volmer equation t0/t = 1 + kq.t0. [Q] 3O2 19 O2-(KO) 2 Indeed, in solutions, those oxygen probes are also described to be deactivated by several reactive oxygen species. One can found several papers in solutions about oxygen, nitric oxide, other stable nitrogen radical such as the intensively used Tempo but also hydrogen peroxide. Mainly because I was convinced that the deactivation by those compounds as in the case of oxygen was occurring because of the paramagnetic nature of the molecule, I recently confirm in solutions that the fluorescent lifetimes of the probes were decreased by only free radical species because they are radical compounds but not by hydrogen peroxide that is not. The quantification of the quenchers can be obtained via the Stern-Volmer equations and the experiments were performed in various solvents in order to stabilize the radical compounds or to make the probe soluble. We can observe that the hydrogen peroxide is not itself detected by neither of the probes. But that all the other free radicals tested are detected, according to diffusion and charge effect. 4.5 17 4 15 Micelle DMSO 3.5 13 3O2DMSO 3O2ethanol 3 11 t0 / t t0 / t NO.(tempo) 9 2.5 7 2 O2-(KO) 2 5 NO. 1.5 3 1 1 H2O2 0.5 1 1.5 2 4 6 8 Quencher Concentration (mM) Quencher Concentration (mM) Tris(2,2'-bipyridyl)ruthenium(II) 1- Pyrene Butyric Acid

5 In living cells Addition of H2O2 solution
Cancerous cell lines 140 150 160 170 180 190 200 210 10 20 30 40 Time (minutes) Lifetime (ns) T. Tharass et al., Analytical Biochem., 2006 Addition of H2O2 (1 mM) CCRF-CEM Human Lymphoblasts 140 Control cells 150 So I will continue with an other experiment that we performed on living cells. But this one was achieved on a level of minutes and not days. In this experiments we again increased hydrogen peroxide but directly though an extra-cellular addition. On the left, in orange, I show the data obtained on control cells. The cells were put under a nitrogen flow at the beginning of the experiment. This was done in order to obtained through the Stern-Volmer equation the intrinsic concentration of ROS without the influence of oxygen concentration. I have to admit that we were quite lucky with this cell lines than is very stable under reduce oxygen pressure. Indeed, we obtain after ten minutes a constant level of ROS, and that, during at least one hour without variation of the energetic state of the cells. But in general we try to perform the experiments within 40 minutes. On the right, after addition of hydrogen peroxide, we observed an increase of the detection of oxygen species. This increase least for twenty minutes and the detection is due to oxygen (for the ten first minutes), induced ROS up to 20 minutes and intrinsic ROS. We found back the level of intrinsic ROS after 20 minutes under nitrogen. With Lower concentrations we don’t detect the ROS changes, with higher concentrations, the number of dead cells through necrosis is too high to perform the experiment. Of course we are not suppose to detect directly the hydrogen peroxide but probably the free radical that are produced by by-reaction of H2O2 in the cells. 10 minutes under Nitrogen atmosphere 20 minutes under Nitrogen atmosphere 160 170 180 Lifetime (ns) 190 200 210 10 20 30 Time (minutes) Fluorescent probe: 1- Pyrene Butyric Acid (PBA) - OXYGEN - INTRINSIC ROS INDUCED ROS -

6 Detection of Oxygen and Reactive Oxygen Species in living cells
CCRF-CEM Human Lymphoblasts 10 (167ns) (206ns) Control cells 160 (173ns) (189ns) 9 198 (210ns) Addition of Inductor If it was a new method for detection of ROS within living cells that we have developed, it must be possible to change the intrinsic level of ROS either by increasing the production of ROS either by inhibiting this production. That is what I present in this slide. The experiments were performed with the pyrene probe. We worked with cell populations because of the heterogeneity of the cell population. This heterogeneity increase the standard deviation compared with experiments in solutions. Moreover, as you can see the differences between each treatment are not so important. For example on this histogram, we double the ROS concentrations in the case of the inductor here in green compared to the control cells in blue. As inductor, we used the adryamicin. It is a chemotherapeutic drug that is known to induce ROS production and you can have a look at the behavior of cells treated with this drug on the poster A04. As inhibitor, here in orange, we used the cysteamin that at the concentration used is indeed an anti-oxidant. We also suppose that when the cells are fixed, for example with a formol solution that keep the integrity of the membrane and still allow the probe loading, the ROS production will be stopped. And because of the and as the reactive oxygen species are indeed reactive, they will not anymore be detected in the cells when the measurements are performed. That what you can observe here in pale yellow. In fact we firstly start to work under nitrogen atmosphere and those measurements were first performed under nitrogen atmosphere. Why to work under nitrogen atmosphere, I didn’t wrote the Stern-Volmer equation here, but the lifetime of the probe depends on the quencher concentrations and if in the cells we have free radical molecules and oxygen that deactivate the probe, it will be difficult to separate the influence of (either of) them. If we reduce the oxygen pressure, we though we will obtain the influence of the reactive oxygen species alone. On the histogram for measurements performed under nitrogen atmosphere we obtained a similar repartition/distribution of the data. We can consider that for fixed cells the ROS concentration as well as the oxygen concentration is nil/zero. So the arrow shows that the ROS production increases when the lifetime values are lower. (151ns) 8 7 Inhibitor Addition 6 Nombre de cellules 5 4 Fixated cells (without ROS) 3 2 1 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 PBA Fluorescent Lifetime (ns) O2 and ROS ROS O2 Experiments under air atmosphere Experiments under nitrogen atmosphere

7 ROS Detection in muscle cells and biopsies
Experiments under air atmosphere (6.2) (11) (4.9) (7.1) (4.6) 20 C: (4.7) C: (4.6) 15 Cells Number 10 In fact it was more complicated when we start to do experiments in primary cells line. The cells were separated from biopsies and the myoblastes were cultivated. Our project is to confirm if the disease we work with, is related to oxidative stress. The muscle cells are not as nice/convenient as our previous cancerous lymphoblast cell lines and it was impossible to performed experiments without oxygen since the cells do not survive long enough under nitrogen flow. The cells coming from healthy people show similar ROS concentrations. For clarity I drew their areas in red. For the cells coming from diseased people we do not really observed a shift of the population though higher ROS concentration. But we observed a broadening of the values with ROS concentration sometimes double. This experiments were performed on myoblastes culture, so in fact, earlier than the morphometric changes observed on myotubes/fiber. We are also adapting (to our apparatus) multiple labeling to increase the number of useful parameters. This was done by changing the filters on the emission path after the microscope. We can obtained with the same filter the lifetime of the pyrene probe and the intensity of NAD(P)H that give an information about the energetic state of the cell. With a second filter we can observed the intensity of the Rhodamine probe that give information about the activity of the mitochondria. This allows using multi-parametric analysis and having a suitable separation of the different cells: most of them, indeed, behaving as the control cells in red. 5 130.00 145.00 160.00 175.00 PBA Fluorescence Lifetime (ns) - Cells activity through NAD(P)H fluorescence intensity (intrinsic fluorescence). - Mitochondria activity trough Rh123 fluorescence intensity. FACTORIAL ANALYSIS -- F1 axe (84.98 %) --> -- F2 axe (12.11 %) -->

8 Home-build Lifetime Apparatus
Image Analyzer Finally I will present you, the homebuilt apparatus we are using to perform these experiments. It is quite simple one with a nitrogen laser, that mean an excitation in the UV and a 3ns impulse. This is quite sufficient for the lifetime of hundred nanosecond showed by oxygen probe such as pyrene derivative and ruthenium complexes. The light arrived on the sample through the microscope and the emitted photon are collected either on the camera and we can observed the microscopic field and selected a single cell then commute on a photomultiplier connected to a fast numerical oscilloscope. The fluorescence decay obtained on the oscilloscope is transferred on the computer. The optical filters, I talk about on the previous slide, are put here before the photomultiplier. This short presentation of the apparatus is not completely without interest. I would like that the method will be accessible to many and specially in the case of biospie we have problem to work with fresh one because of the distance between the only apparatus and the people interested. I will be interested that the experiments will be confirmed on a commercial apparatus. Such an apparatus need sensitivity as the pyrene probe show an intensity of one ten compared to the intrinsic fluorescence due to NAD(P)H at the concentration used. We also need a low level of irradiation as the cells functions will be affected by high irradiation. We even reduced the laser power with neutral filters in order to obtain reproducible decay on a same cells. That will certainly forbid the use of single photon counting apparatus. 0.002 0.004 0.006 0.008 0.01 200 400 600 800 1000 Numerical Oscilloscope PulsedLaser Camera Photo-Multiplicator

9 In Progress Cell model Anti-oxidants Inside the cell
Cells cultivated under 2% of oxygen (posterA04). Measurements on biopsies fresh versus frozen. Anti-oxidants ROS influence in various diseases Afin de regarder ce qui se passe lorsqu’on voit des différences morphologique, j’ai donc commencer les mesures sur les cellules organisées en myotubes. Nous envisageons aussi de cultiver les cellules sous 5 % d’oxygène, qui est une concentration plus proche de ce qui est réellement présent dans les muscles et de mettre au point des mesures directement sur les biopsies. Il faudra de plus adapter la méthode en normoxie, afin d’obtenir des valeurs pour les variations de ROS, ce qui permettra alors d’élargir le champs des applications. Pour cela il faudra mieux connaitre la diffusion de l’oxygène dans la cellule. La diffusion compense-t-elle la diminution de l’oxygène utilisé dans la chaîne oxydative et cette diffusion de l’oxygène est-elle assez rapide pour compenser cette perte? Les comparaison entre les essais sous air et sous azote laisse penser que la diffusion de l’oxygène est effectivement plus rapide que les phénomènes lié aux ROS que nous étudions. En conclusion, c’est donc à des modifications de la physiologie de la cellule vivante que nous nous intéressons, modifications pouvant être liées à des pathologies mais aussi être des réactions à des stimuli environnementaux. Nous espérons un développement tant au niveau des connaissances fondamentales des diverses maladies que nous serons amené à étudier que pour l’aide au diagnostic et au traitement des patients. Merci, je viens vous présenter une méthode pour la détection de l’oxygène et des espèces réactives de l’oxygène dans les cellules vivantes. Les mesures de durées de vie de fluorescence dont je vais vous parler sont utilisées depuis de nombreuses années pour la détection de l’oxygène mais il semblerait que très peu de personne n’ai mis à profit leur sensibilité aux espèces réactives de l’oxygène. Quelques essais en solution ont été faits, mais c’est une méthode nouvelle que je vous présente et elle a été mise au point sur des cellules vivantes en culture et nous espérons montré son fonctionnement sur du matériel cellulaires intacts tel que les biopsies. Cytosol H2O O2 O2 externe ROS Inside the cell Probes for the mitochondria

10 Acknowledgments Oxygen et ROS Myopathy
Ligue contre le cancer, comité départemental des Pyrénée Orientale et du gard. Jean-Marie Salmon, Jean Vigo (Images, Perpignan), Tareck Rharass (Ph.D student) Oslem Oter (University of Dokuz Eylul, Turkey) Myopathy Dalila Laoudj, Gilles Carnac, Marietta Barro (CRBM/CNRS, Montpellier), Jacques Mercier, Delphine Delample (CHU Arnaud de Villeneuve, Montpellier) I would like to thank my close colleagues jean Vigo and jean- marie Salmon. And specially, tareck rharras, who is in his last year of its pHD and work on the cancer proposal. I would like to emphasizes a last time of the difficulty to have fresh biopsies if we do not develop a tradable apparatus.

11 Mono-exponential decay
Lifetime recorded on isolated cells 0.02 0.04 0.06 0.08 0.1 0.12 200 400 600 800 1000 Temps (ns) Signal de sortie (mV) PBA NAD(P)H Bi-exponential decay Lorsque nous faisons les mesures de ddv sur ces cellules isolées, voici ce que nous obtenons expérimentalement ici avec le filtre à 404 nm. Ici nous avons un exemple de déclin obtenu pour une cellule vivante isolée non chargé en sonde. Le déclin du à la fluorescence propre de la cellule autour de 400 nm est attribuée aux molécules de NAD(P)H. Chargé avec la sonde à une faible concentration, le déclin est manifestement double, la première partie courte due à la fluorescence intrinsèque de la cellule, la deuxième partie correspond au groupement pyrène. Il est facile à partir des durées de vie très différentes de séparer les paramètres correspondant aux PBA et NAD(P)H qui renseigne sur le fonctionnement de la cellule. Afin de travailler sur un déclin plus simple, nous avons choisit de soustraire ces deux déclins entre eux. On modélise ce déclin et on obtient une valeur, la durée de vie de la sonde, associés aux quantité de molécules d’oxygène et des espèces RO. Mathematical Treatment 0.000 0.005 0.010 200 400 600 800 1000 Temps (ns) Signal de sortie (mV) Mono-exponential decay