In lignocellulosic biomass the activity of enzymes analyzing polysaccharides is limited by the existence of non-specific interactions with lignin. FRET analysis is a relevant approach for evolution at the nano scale. Obtaining all sections after can be a challenge depending on the biomass.
Take your time doing the sectioning with the microtome and perform gentle washings. Collating fluorescent lifetime and spectrum measurements allows a quantitative sensitive and unambiguous FRET determination between lignin and TAG molecules in situ. This method employs the use of tricky techniques, like sample sectioning on multidimensional microscopy acquisition and analysis that can be easy to learn with a visual demonstration.
To prepare plant samples from wood, wheat, fibers, or fragments use razor blades to cut one centimeter long samples without damaging the structure of the material and place the samples under vacuum to remove any air. Submerge the samples for 24 hours in successive 30%and 50%concentrations of PEG diluted in water at room temperature with gentle stirring. Followed by 24 hours submerged in a 100%concentration of PEG at 70 degrees Celsius.
After the 100%PEG immersion, place the samples in capsules on a 70 degree celsius hotplate and progressively decrease the plate temperature in five degrees celsius increments until the plate reaches room temperature. When the samples have cooled, use a microtome and disposable blades to carefully obtain flat 30 to 60 micrometer thick sections from each PEG block. Remove the PEG from the sections with three five minute gentle water washes.
After the last wash, incubate up to three sections per tube with 500 microliters of freshly prepared fluorescent probe for 72 hours protected from light. At the end of the staining incubation, use a brush to transfer the sections to a slide before mounting the sections between a glass slide and a number 1.5H cover slip. To determine the spectral window width of the sFLIM detector, place urea crystals in a culture box with a 0.17 micrometer glass bottom and place the box on a microscope sample holder.
Select the 20X objective and a titanium doped sapphire laser with a 900 nanometer wavelength and 2%power and turn on the scanning. To perform sFLIM measurements, switch the system to sFLIM Mode. To collect the second harmonic signal on the sFLIM detector, gradually adjust the laser excitation wavelength from 900 to 980 nanometers until the second harmonic signal is collected in the second spectral channel.
Then determine the spectral window length. Switch to the nonD scanned mode to send fluorescent photons to the sFLIM detector. Set the sFLIM Acquisition to the Enable Mode to allow photon counting on the single photon counter.
When the instrument is ready, place a control native wheat straw alone plant section between the slide and the cover slip on the microscope stage and set the system to Continuous Mode to allow the laser scan. Set a 30 second time collection and check that the constant fraction discriminator is between one times 10 to the fourth and one times 10 to the sixth. Then select the measurement area on the sample and click Start.
At the end of the measurement, save the sdt file and repeat the measurement for at least nine more samples. A balance must be found between achieving enough collected photons for the photon while avoiding pulse buy up and photo induced effects that can alter the florescence lifetime. Top analyze the acquired sFLIM data, import the native wheat straw alone data files into the single photon counter software and open Option and Mode to select Incomplete Multi Exponential 12.5 nanoseconds.
Under Options and Preferences, select Calculate Instrumental Response Automatically and select the To Exponential Fit Model. For each channel, apply the fitting model and save the fit parameters to a spreadsheet. For a comparison between the channels and experiments calculate the main fluorescence lifetime in a spreadsheet.
To calculate the mean lifetime for at least 10 samples in all of the channels, analyze the main fluorescent lifetime on the donor channels to determine the dedicated channel value that chose the highest photon number and corresponds to the lignin maximum emission. To compare the sFLIM and EFRET values between the control native wheat straw sample and an experimental sample, consider a positive EFRET associated with a homogenous lifetime decrease between the control and the experimental samples to be analyzed as a FRET event. In this representative experiment, sFLIM curves show some of the modifications that can be achieved between the reference sample and the two interaction cases.
Indeed, the fluorescence increase in spectral regions corresponding to the Rhodamine emission range are easily visualized. Careful observation of the three first channel photon decay curves, also reveals a stronger inflection for native wheat straw with Dextrin tagged with Rhodamine, than for native wheat straw incubated with PEG tagged with Rhodamine. After fitting the photon decay curves and calculating each channel main fluorescence life time, the FRET signature becomes more obvious.
For example, while the fluorescence life time alternatively increases and decreases along the fluorescence spectrum for the native wheat straw sample, a clear FRET signature is observed for both native wheat straw sections incubated with PEG tagged with Rhodamine and native wheat straw with Dextrin tagged with Rhodamine samples. In the context of lignocellulosic biomass hydrolysis, this method can easily be transferred to enzyme interaction studies in various biomass samples requiring a rigorous characterization of each new biomass. Since sFLIM does not require fixation of the sample it paves the way for dynamic enzyme lignin interaction studies and is likely to guide enzyme engineering strategies and biomass pre-treatments.
As most time, life time measurements require the use of pulse infrared laser sources. The appropriate safety precautions must be taken accordingly.
