The overall goal of this procedure is to directly image and compare lignification in plant cell walls with spatial resolution that has sub micrometer without staining or labeling of the samples in a close to native state. This is accomplished by first cutting thin sections from the native tissue by microtone. The next step is to acquire a spectral map of the region of interest of the sample in the confocal Raman microscope.
This is accomplished by Rasta scanning the sample and recording a ramen spectrum for each sample position. The final step is to analyze the spectral dataset and to compute chemical images where the spatial distribution of lignin is visualized by integrating the intensity of specific ramen bands, which are attributable to molecular vibrations of lignin. Ultimately, results can be obtained that show the spatial distribution of lignin without staining or labeling of the samples.
Through confocal rama microscopy, the lignin signal intensities can be compared between different measurements, tissues, samples, and species. Hi, I'm Jim Shock, a staff scientist at the Molecular Foundry at Lawrence Berkeley National Lab. Hi, I'm Martin Schmidt from the Energy Biosciences Institute at the University of California Berkeley.
Today we're gonna show you a procedure for the label free imaging of lignification in plant cell walls. So let's get started. To begin this procedure, place the plant sample such as poplar stem wood or Arabidopsis ANA stem In the microtome here, Arabidopsis ANA stem sections are prepared.
Then cut thin sections, typically 20 microns thick from the native tissue. For best results, the sample should be hydrated when finished. Cutting, transfer each plant section onto a glass microscope slide.
Finally, soak the plant section in de to rated water or D two O cover with a glass cover, slip and seal onto the microscope slide to prevent evaporation of D two O.D two O is not native to the sample and is useful to perform intensity normalization. The plant sections are now ready for imaging or they can be stored for future use. Before staging the sample, apply immersion oil to the microscope objective and the cover slip place and secure the microscope slide on the piezoelectric scan stage of the microscope with the cover slip facing the microscope objective, using a high numerical aperture immersion microscope objective, view the sample through the cover slip and locate the sample area of interest.
Next, switch off all other laboratory and microscope light sources. Perform position resolve micro spectroscopic measurements by focusing band pass filtered monochromatic green light from a CW laser onto the sample. With a typical power of 10 to 30 milliwatts autofluorescence can occur in some samples, which may prohibit useful measurements.
If this is the case, then attempt excitation with a longer wavelength laser light. The back scattered stoke shifted. Ramen light is collected by the microscope.
Objective passes through a dichroic mirror, a pinhole, which serves as a spatial filter in the confocal setup and a long pass filter. The light is focused into the slit of a grating spectrometer where it is spectrally dispersed and detected by a cord CCD camera giving a Raman spectrum for chemical imaging and visualization of the spatial lignin distribution. Raster scanning is performed by moving the sample through the laser focus with the Piso electric scan stage and recording a Raman spectrum for each sample position.
Raster scanning will generate a two dimensional spectral map of the sample to generate three dimensional spectral maps. Step the laser focus consecutively along the Z direction, and then stack these two dimensional maps. Follow up the data acquisition with analysis.
To visualize the lignin distribution for chemical imaging and lignin visualization, the collected data are analyzed using matlab. The data are arranged in a three dimensional hyperspectral cube, which is composed of the two spatial dimensions, and a third dimension for the spectral signals for the lignin analysis. A spectral region between 1, 550 and 1, 700 reciprocal centimeters is considered.
The spatial distribution of lignin is visualized by integrating the intensity from 1, 550 to 1, 700 reciprocal centimeters of the baseline corrected spectra. An alternative to baseline correction is computing the second derivative spectra and using the second derivative peaks for analysis. Further analysis of lignin localization and chemistry, especially with regard to confer aldehyde and conferral alcohol moieties can be carried out as well by fitting the three bands between one thousand six hundred and one thousand seven hundred reciprocal centimeters to gian peaks and evaluating the area under the peaks intensity.
Normalization between different spectral maps is a critical step, which allows one to compare lignin signal intensities between different measurements, tissues, samples, and species. To do this, obtain average lumen spectra through K means clustering classification, and use the peak height of the extrinsic OD stretching band. Found around 2, 500 reciprocal centimeters in the average lumen spectra.
As a reference, careful sample preparation gives suitable plant tissue sections high quality and spatially resolved. Raman spectra of, for example, poplar stem wood in derated water are obtained using ramen microscopy. The shaded area of the spectrum, which is expanded here marks the spectral region with the three peaks specifically attributable to lignin.
By integrating the ramen signal intensity from 1, 550 to 1, 700 reciprocal centimeters, a ramen lignin image can be obtained. We've just shown you how to image lignification in plant cell walls using confocal ramen microscopy. Thanks for watching and good luck with your experiments.
