The overall goal of this experiment is to characterize diffusional motion of topological polymers, especially cyclic polymers, under entangled conditions at the single molecule level. This method can help answer key questions in the field of polymer physics, such as topology-dependent spacio-temporal dynamics of polymers. The main advantage of this technique is that heterogeneous diffusion can be quantitatively characterized at the single-chain level, which is usually hidden behind on some level.
To begin this procedure, dissolve perylene diimide salt in 150 milliliters of water, then dissolve monofunctional poly(THF)in four millimeters of acetone. Add the acetone solution drop-wise to the vigorously stirred aqueous solution. Collect the formed precipitate by vacuum filtration.
Next, prepare a five milligram per milliliter solution of the precipitate in toluene. Reflux the solution for four hours. After allowing the solution to cool, completely remove the solvent under reduced pressure by rotary evaporation.
When finished, dissolve the residue in a 2:1 mixture of n-hexane:acetone and filter the resulting solution through a plug of silica gel. Then add the filtered solution to ice cold water to precipitate the product. To prepare the polymer melt sample, add 100 microliters of non-labeled linear poly(THF)to a glass bottle and heat it to approximately 25 degrees celsius using a hair dryer.
Prepare a 10 to the minus six molars solution of the fluorophore incorporated polymer solution in chloroform. Then add one microliter of this solution to 100 microliters of the non-labeled linear poly(THF)melt. After thoroughly mixing the sample with a pipette tip, evaporate the chloroform by heating with a hair dryer.
Using a micro-pipette place 10 microliters of the sample on a cleaned cover slip. Place another cleaned cover slip on the sample and gently press the two cover slips together using plastic tweezers. Next, attach an objective heater to the objective lens of an inverted microscope and set the temperature to 30 degrees celsius.
Place one drop of immersion oil on the lens and mount the sample on the microscope stage. Ensure that a sample thickness of approximately 10 micrometers is obtained by checking the axial position of the bottom and top surface of the sample. Then adjust the focus of the microscope to a few micrometers above the bottom surface of the sample.
Following this, apply an electron multiplying or EM gain to a CCD camera in order to obtain a high quality fluorescent image of the single fluorophore. Now set a region of interest using the software controlling the camera. To optimize the experimental conditions, adjust the illumination area of the sample to approximately 20 micrometers in diameter using the diaphragm inserted in the excitation beam path.
Set the excitation laser power at the sample to four to eight milliwatts by manually selecting an appropriate neutral density filter inserted in the excitation beam path. Finally, record 500 to 1000 fluorescence image sequences of the fluorophore incorporated polymer in the melt state at a 100 to 200 hertz frame rate. Time-lapse single molecule florescence images were measured for the four-armed and eight-shaped polymers and show spatially isolated bright and sharp spots due to the incorporation of the highly fluorescent perylene diimide fluorophore into the chains.
The frequency histograms of the diffusion coefficient determined by mean-squared displacement analysis display broad distributions resulting from both the statistical error of the analysis and heterogeneity of the diffusion. The frequency histograms show clear deviations from the homogeneous diffusion model, which demonstrates heterogeneous diffusion of the polymer molecules. The single and double Gaussian models hit the experimentally obtained cumulative distribution functions well and demonstrate that the diffusion of the formed polymer is described by the broad distribution of the diffusion coefficient, whereas the eight-shaped polymer displays two distinct diffusion modes.
Once mastered, the synthesis of polymers can be done in six hours and the single molecule immersion experiment can be done in several hours if they are performed properly. After it's development, this technique paved the way for researchers in the field of polymer science to explore polymer dynamics in crowded environments. After watching this video, you should have a good understanding of how to characterize diffusive motion of topological polymers under entangled condition at the single-chain level.
Don't forget that working with lasers and organic solvents can be extremely hazardous and precautions such as laser safety and review of the MSDS should always be taken while performing this procedure.
