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- Alrifaiy, Ahmed, et al.
(author)
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Development of microfluidic system and optical tweezers for electrophysiological investigations of an individual cell
- 2010
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In: Optical Trapping and Optical Micromanipulation VII. - Bellingham, Wash : SPIE - The International Society for Optics and Photonics. - 9780819482587
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Conference paper (peer-reviewed)abstract
- We present a new approach of combining Lab-on-a-chip technologies with optical manipulation technique for accurate investigations in the field of cell biology. A general concept was to develop and combine different methods to perform advanced electrophysiological investigations of an individual living cell under optimal control of the surrounding environment. The conventional patch clamp technique was customized by modifying the open system with a gas-tight multifunctional microfluidics system and optical trapping technique (optical tweezers).The system offers possibilities to measure the electrical signaling and activity of the neuron under optimum conditions of hypoxia and anoxia while the oxygenation state is controlled optically by means of a spectroscopic technique. A cellbased microfluidics system with an integrated patch clamp pipette was developed successfully. Selectively, an individual neuron is manipulated within the microchannels of the microfluidic system under a sufficient control of the environment. Experiments were performed to manipulate single yeast cell and red blood cell (RBC) optically through the microfluidics system toward an integrated patch clamp pipette. An absorption spectrum of a single RCB was recorded which showed that laser light did not impinge on the spectroscopic spectrum of light. This is promising for further development of a complete lab-on-a-chip system for patch clamp measurements.
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| 2. |
- Alrifaiy, Ahmed, et al.
(author)
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Ett mikroflödessystem för multipla undersökningar av enstaka biologiska celler under hypoxiska förhållanden
- 2011
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Conference paper (peer-reviewed)abstract
- Introduktion: Syftet med studien är att studera enstaka nervcellers respons vid syrebrist i ett mikroflödessystem för att förstå nervcellens respons vid stroke. Målet med studien var att utveckla ett slutet mikroflödessystem som ger optimal kontroll av den omgivande miljön och samtidigt möjliggöra elektrofysiologiska undersökningar under kontrollerade syreförhållande. Material och metoder: Mikroflödescellen utvecklades för ett inverterat mikroskop, utrustad med en optisk pincett och optisk spektroskopi samt patch-clamp för elektrofysiologiska studier på en enstaka nervcell. Istället för att föra en pipett mot en cell i ett öppet system fångades en enskild cell optiskt i ett slutet mikroflödessystem och fördes mot en fixerad patch-clamp mikropipett. Cellen utsattes för olika syrehalter och övervakades av ett UV-Vis spektroskop medan cellens elektrofysiologiska aktivitet registreras med patch-clamp. Det slutna mikroflödessystemet med integrerad mikropipett, kopplades till ett pumpsystem för införandet av celler och buffert med olika kemiska egenskaper och syrehalter. I ett inverterat mikroskop integrerades optisk pincett, UV-Vis spektrometer och patch-clamp. Resultat och diskussion: För att pröva konceptet fångades och fördes en röd blodcell optiskt mot mikropipetten som befann sig på en fast position i mikroflödescellen. Cellens syrebindningstillstånd varierades genom att tillsätta syrefri eller syresatt buffert och registrerades med UV-Vis spektrometern. I ett vidare experiment manipulerades en nervcell optiskt i ett öppet system mot patch-clamp pipetten och elektrofysiologiska mätningar utfördes. Vi kunde verifiera att den optiska pincetten inte påverkade den elektrofysiologiska mätningen. För närvarandet utförs elektrofysiologiska mätningar i det slutna mikroflödessystemet för att se hur nervcellerna reagerar under varierande syrehalt. Genom mätningarna hoppas vi att få mer kunskap om försvarsmekanismerna som igångsätts av neuroner under syrefattiga förhållanden.
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| 3. |
- Alrifaiy, Ahmed, et al.
(author)
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Hypoxia on a chip - a novel approach for patch-clamp studies in a microfluidic system with full oxygen control
- 2012
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In: World Congress on Medical Physics and Biomedical Engineering, May 26-31, 2012, Beijing, China. - Berlin : Encyclopedia of Global Archaeology/Springer Verlag. - 9783642293047 - 9783642293054 ; , s. 313-316
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Conference paper (peer-reviewed)abstract
- A new approach to perform patch-clamp experiments on living cells under controlled anoxic and normoxic conditions was developed and tested. To provide an optimal control over the oxygen content and the biochemical environment a patch-clamp recording micropipette was integrated within an oxygen tight poly-methyl methacrylate (PMMA) based microchip. The oxygen content within the microfluidic chamber surrounding patch-clamp micropipette was maintained at 0.5-1.5 % by a continuous flow of artificial extracellular solution purged with nitrogen. The nerve and glial cells acutely obtained from the male rat brain were trapped by the optical tweezers and steered towards the patch-clamp micropipette through the channels of the microchip in order to achieve a close contact between the pipette and the cellular membrane. The patch-clamp recordings revealed that optical tweezers did not affect the electrophysiological properties of the tested cells suggesting that optical trapping is a safe and non-traumatizing method to manipulate living cells in the microfluidic system. Thus, our approach of combining optical tweezers and a gas-tight microfluidic chamber may be applied in various electrophysiological investigations of single cells were optimal control of the experimental conditions and the sample in a closed environment are necessary.
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| 4. |
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| 5. |
- Bitaraf, Nazanin, et al.
(author)
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Development of a multifunctional microfluidic system for studies of nerve cell activity during hypoxic and anoxic conditions
- 2009
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In: World Congress on Medical Physics and Biomedical Engineering. - Berlin : Springer Science+Business Media B.V.. - 9783642038976 ; , s. 176-179, s. 176-179
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Conference paper (peer-reviewed)abstract
- Hemoproteins usually supply cells and tissue with oxygen. A new hemoprotein mainly present in nerve cells called Neuroglobin was recently discovered. Enhanced expression of the protein has been shown to reduce hypoxic neural injury but the mechanism behind this function remains unknown. Methods enabling investigation of the protein in single functional neurons need to be developed. Here, we have studied how the electrical signaling capacity of a neuron was affected by hypoxic environments. Preliminary results show a trend of higher noise-level when a neuron is exposed to hypoxic compared to normoxic surroundings, which implies increased ion-channel activity. The setup used today shows shortages such as reduced control over the oxygen content due to leakage. Therefore, a gas-tight, multifunctional microfluidic system is under development which enables us to study influences of Neuroglobin concentrations on neuronal activity during hypoxia and anoxia. For electrophysiological recordings a patch-clamp micro pipette will be molded into the walls of the microfluidic system. A single biological cell is steered towards the pipette and attached there by means of optical tweezers. The Neuroglobin oxygen binding state will be studied using optical spectroscopy and the neuron environment will be manipulated by applying flows of varying oxygen content through the microfluidic system. This system will constitute a powerful tool in the investigation of the Neuroglobin mechanism of action.
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| 6. |
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| 7. |
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| 8. |
- Bitaraf, Nazanin
(author)
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The electrophysiological response of medial preoptic neurons to hypoxia and development of a system for patch-clamp measurement with full oxygen control
- 2011
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Licentiate thesis (other academic/artistic)abstract
- A stroke is caused by interruption of the blood supply to the brain. Yearly 15 million people around the globe endure a stroke and the costs and suffering for the people involved and the society are immense. The aim of this thesis was to investigate the response to oxygen deprivation in neurons from the medial preoptic nucleus (MPN) that have a high abundance of neuroglobin. The long term goal is to investigate the neuroprotective role of the protein in relation to stroke. Initially, the electrophysiological response of neurons to hypoxic exposure in an open system was assessed with a conventional patch-clamp setup. The first aim was to see how well the conventional system worked and if it needed improvement. Secondly, the MPN had never been investigated regarding oxygen, deprivation; hence the electrophysiological response under hypoxia needed to be investigated. The conventional patch-clamp system only allowed a reduction of the oxygen content to a level of 3-6% but not total control of the cell environment. The medial preoptic neurons showed mainly an increase of their resting membrane potential at hypoxia. The voltage activated Ca2+ and K+ currents displayed a clear attenuation when cells were subjected to hypoxia. Non-L-type Ca2+ channels were affected by hypoxic exposure and one cell indicated participation of Ca2+ activated K+ channels. However, a response could only be seen in approximately fifty percent of the neurons in the open system. This may have been due to the fact that full control of the oxygen around the neurons at hypoxia could not be achieved. A new system with full control of the ambient oxygen had to be developed in order to investigate this. After the conclusions of the first experiments, a system was developed were a labon- a-chip system was combined with the patch-clamp technique. A microfluidic system with a patch-clamp micropipette integrated was combined with optical tweezers for 3D maneuvering of the neurons. The development of patch-clamp in combination with a microfluidic system and optical tweezers allowed for full oxygen control. The experiments showed that the electrophysiological measurements were not affected by the laser when an infrared laser was used. The microfluidic system allowed very good oxygen control reaching levels of 0.5-1.5 % compared to 3-6 % in the open system. In summary, this work suggests that high voltage activated Ca2+ channels, and K+ channels are involved in the hypoxic depolarization of medial preoptic neurons. Full control of ambient oxygen in cell vicinity could be achieved by the combination of microfluidics, patch-clamp and optical tweezers. The results can be used in future studies to better understand the reaction of the brain to oxygen deprivation caused by stroke.
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