The overall goal of this experiment is to present a general method for visualizing lignin, cellulose, and hemicellulose in the plant cell wall by Raman imaging and multivariate analysis. This method can help answer key questions in the plant biomass field, such as how do the major chemical components distribute in the plant cell wall. The main advantage of this technique is that labor-free and non-destructive information about the sample can be achieved with minimal sample preparation.
To begin, cut a small tissue block from the plant sample. Immerse the tissue in boiling deionized water for 30 minutes. Then, immediately transfer it to deionized water at room temperature for 30 minutes.
Repeat this step until the tissue sinks to the bottom of the container, indicating that the air in the tissue has been removed and that the tissue has softened. Prepare 20%50%70%and 90%aliquots of PEG and deionized water, as well as pure PEG. Keep the solutions a 65 degrees Celsius.
Incubate the tissue in a series of graded PEG baths to displace the water, and allow the PEG to infiltrate. Dry in 20%PEG for one hour, 50%PEG for one and a half hours, 70%PEG for two hours, 90%PEG for two hours, and 100%PEG for 10 hours. Next, pre-warm a cassette at 65 degrees Celsius in an oven.
Pour the tissue block containing PEG into the cassette, and then place the tissue block in a desired position using pre-warmed tweezers or needles. Slowly cool down the cassette, and store the tissue at room temperature until use. Dissect the PEG block containing the target tissue into a small block using a sharp razor blade.
Mount the small PEG block onto the microtome, and cut thin sections from the PEG block. Then, rinse the sections with deionized water in a watch class 10 times to remove the PEG from the tissue. Next, immerse the sections with toluene and ethanol for six hours to remove the extractives.
Prepare the reaction liquid by mixing 65 mL of deionized water, 0.5 mL of acetic acid, and 0.6 g of sodium chloride in a beaker. Add one section and 3 mL of reaction liquid to a 5 mL vial. Screw on the top of the vial.
Then, heat the vial in a water bath at 75 degrees Celsius for two hours to remove the lignin of the tissue. Rinse the section with deionized water in a watch glass 10 times. Next, transfer the untreated and de-lignified section to a glass microscope slide.
Unfold the section carefully using brushes or needles. Remove the extra deionized water with tissue paper. Then, immerse the section in deuterium oxide.
Cover the sample with a glass cover slip. Seal the cover slip with nail polish to prevent the evaporation of the deuterium oxide. Open the instrument operating software of the confocal Raman microscope.
Turn on the laser, and focus on the service of the crystal and silicon with a 100x microscope objective. Click the calibration button to calibrate the instrument. Switch the instrument to the optical microscope mode and turn on the microscope lamp.
Mount the microscope slide on the stage, with the cover slip facing the objective. View the sample with the 20x microscope objective and locate the area of interest. Switch to the immersion microscope objective.
Apply immersion oil to the cover slip, then focus on the surface of the sample. Next, switch the instrument to the Raman testing mode and turn off the microscope lamp. Chose a mapping area by using a rectangular tool.
Alter the step size to determine the number of obtained spectra. Be aware that the step size should be larger than the spot diameter, calculated by the numerical aperture of the objective. Sizes below this will result in over-sampling.
Set the optimum spectral parameters to obtain the best signal-to-noise ratio and spectral quality, and an appropriate acquisition time depending on the sample suitability. Generally, input the imaging parameters in the instrument software using a laser wavelength of 532 nanometers, a filter of 100%a hole of 300, a slit of 100, a spectrometer of 1840 inverse centimeters, grooves of 1200T, a 60x oil objective, and an acquisition time of two seconds. Save the spectral data before data processing, and convert the data to a universal format before proceeding to data analysis as described in the text protocol.
The original Raman spectra of the poplar cell wall are shown here. Two major noise signals, including baseline drifts and cosmic spikes, spill over into the channels along with the actual signals. These should be removed prior to data analysis.
A noise reduction technique such as the Savitzky-Golay algorithm or the wavelet algorithm can be applied to eliminate these noise signals. For lignin imaging, consider the spectral peak around 1600 inverse centimeters due to the aromatic ring symmetric stretching vibration. For polysaccharide imaging, use the spectral peak around 2889 inverse centimeters because of the CH and CH2 stretches.
However, Raman images of cellulose and hemicellulose cannot be generated directly. Delignification is a necessary procedure to expose the spectral characteristics of them. Generally, we are new to this method.
We struggle, because it requires training in handling and appearance of sample sectioning and the data analysis. After watching this video, you should have a good understanding of how to acquire chemical information about the plant cell wall using in-situ methods. Following this procedure, other methods like chemical pre-treatment can be performed in order to answer additional questions, like the recalcitrance of the plant cell wall.
Though this method can provide insight into the internal structural nature and the topochemistry within the plant cell wall, it can also be applied to other biological samples.
