Fusion of Giant Unilamellar Liposomes

 

We demonstrate the fusion of giant unilamellar POPC liposomes brought together by the use of optical tweezers. The liposomes are formed by the electroformation method. Prior to the electroformation process DIOC dye is mixed in with the lipids.  During electroformation DIOC dye attaches to the membranes of the forming liposomes. A solution of sucrose is added to the electroformation chamber and the liposomes enclose this solution as they form. The liposomes are about 10-50 micrometers in diameter. Since it is very hard to optically trap giant unilamellar liposomes the sucrose solution helps increase the trapping ability by changing the index of refraction of the vesicles. After the liposomes are formed they are collected in a flask and mixed in with HEPES buffer. The sucrose solution also makes the vesicles heavier, giant liposomes are collected from the bottom after a few minutes of low speed centrifuge of the flask.

After two liposomes are brought together by dual optical tweezers, the fusion process is initiated by a UV pulse.  We excite the DIOC dye on the membranes of the liposomes and image the process fluorescence microscopy. The polarization of the excitation light is continually varied during the process in order to observe the orientation of the lipids on the membranes, before, during and after fusion. Our main aim is to demonstrate whether a stalk forms during the fusion process or not. Details of our optical setup, computer control and various challenges that we have overcome will be reported.

Authors      :  Erge Edgu-Fry, Rani Kishore, Kris Helmerson, Jeeseong Hwang, PL

Division     :  Atomic Physics

Laboratory :  Physics

Room         :  B247, Bldg 216

Mail Stop   :  8424

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Fax             :  301-975-8272

E-mail        :  erge.edgu-fry@nist.gov

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Physics