Multi scale fluorescence studies: The spinning disk confocal fluorescence lifetime imaging microscopy & single molecule nano-metronome & single molecule study of tetramethyrhodamine -DNA interaction

The thesis is consisted of three different sections independent of each other. The first part involves the frequency domain fluorescence lifetime imaging microscopy (FLIM) and an improvement of FLIM setup to decrease the out of focus background fluorescence without drastically sacrificing the data acquisition speed while taking fluorescence lifetime data. The setup incorporates a spinning disk confocal unit to the conventional wide-field fluorescence lifetime imaging; therefore, it is a marriage between confocality and data acquisition speed. The detailed discussion about the principle of the setup in conjunction to the benefits when the setup is used for imaging thick specimens is presented. In addition, a new concept of using the Wavelets analysis on fluorescence lifetime images is introduced. Besides facilitating morphology recognition and noise reduction usually shown in normal image analysis, the benefit of wavelets analysis on decomposing an image into different spatial frequencies proves to be useful in removing the lifetime information of the background region out of the lifetime information from the small structures of interest. Moreover, the solutions to the artifacts often encountered in frequency domain fluorescence lifetime measurements are shown. Finally, the FLIM setup is used to study the close interactions between structural proteins in the body wall muscle cells of the nematode Caenorhabditis elegans and visualize the activation states of a signaling protein in cultured neurons of Drosophila melanogaster based on the change in the fluorescence lifetime of CFP at different energy transfer efficiencies between the CFP and YFP pair.

In the second part, a nano-device called nano-metronome constructed out of four single stranded DNA is presented on single molecule level. The mechanism of the device is based on the thermally driven fluctuation between two conformations of the Holiday four way junction. Two single stranded overhangs that can form the sticky ends on two arms of the junction are used to change the rates of conformational fluctuations. We are able to show that the rates depend on the number of sticky base pairs in addition to the changes in ionic strength of the solution; therefore user controllable. Moreover, the sticky end can be reversibly deactivated and re-activated if a short single stranded DNA, so called the deactivator, capable of competing with the binding between the two overhangs and another short single stranded DNA, so called the activator, capable of peeling off the deactivator are provided. Since the device displays clearly distinguishable responses even with a single basepair difference, it may lead to a single molecule sensor of minute sequence differences of a target DNA.

The final part presents the single molecule study on a behavior of a popular fluorescent dye called Tetramethylrhodamine (TMR) attached to the end of DNA oligomer. The study reveals that in the absence of any biological activities of DNA, the fluorescence emission of TMR undergoes multiple slow transitions among at least four different fluorescence states. While the actual number of fluorescence states and therefore the actual rates of transition among them remains open for discussion and more control measurements are definitely required, we are able to show that each fluorescence state lasts on the average in a range of hundreds of milliseconds to several seconds. These long dwell times are not observable in most of the studies done on freely diffusing TMR-DNA conjugate. It is unlikely that the fluctuation originates from the non-specific interactions between the dye and the glass surface and the interaction appears to be specific to TMR-DNA system. Therefore, TMR should be used with caution when conjugated to DNA.